WO2022251663A2 - Nouveaux agents viraux anti-arn universels - Google Patents

Nouveaux agents viraux anti-arn universels Download PDF

Info

Publication number
WO2022251663A2
WO2022251663A2 PCT/US2022/031381 US2022031381W WO2022251663A2 WO 2022251663 A2 WO2022251663 A2 WO 2022251663A2 US 2022031381 W US2022031381 W US 2022031381W WO 2022251663 A2 WO2022251663 A2 WO 2022251663A2
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
compound
cycloalkyl
substituted
aryl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2022/031381
Other languages
English (en)
Other versions
WO2022251663A3 (fr
Inventor
Raymond F. Schinazi
Franck Amblard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Emory University
Original Assignee
Emory University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Emory University filed Critical Emory University
Priority to US18/562,911 priority Critical patent/US20240262857A1/en
Publication of WO2022251663A2 publication Critical patent/WO2022251663A2/fr
Publication of WO2022251663A3 publication Critical patent/WO2022251663A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled

Definitions

  • RNA viruses such as Coronaviruses, Picomaviruses, Hepeviruses, Chikungunya fever (CHIK), Ebola, Influenza, RSV, Yellow Fever, Eastern, Western, and Venezuelan Equine Encephalitis (EEE, WEE and VEE, respectively), and Zika virus infections.
  • RNA viruses such as Coronaviruses, Picomaviruses, Hepeviruses, Chikungunya fever (CHIK), Ebola, Influenza, RSV, Yellow Fever, Eastern, Western, and Venezuelan Equine Encephalitis (EEE, WEE and VEE, respectively), and Zika virus infections.
  • NHC has the ability to pair ambiguously as cytidine or uridine, thus introducing an elevated mutation load when incorporated into most RNA.
  • NHC can concentrate in the viral RNA genome over cellular RNA as viral RNA replication passes through the ribonucleotide pool multiple times for the synthesis of both plus and minus strands, resulting in lethal mutagenesis.
  • RNA viruses including, but not limited to, Coronaviridae, such as MERSr-CoV, SARS-CoV-1, SARS-CoV-2, HCoV-OC43, HCoV- 229E, HCoV-NL63, and HCoV-HKU1, Picomaviridae, Hepeviridae, Noroviruses, Zika, Dengue, Mayaro, Influenza A and B, Parainfluenza, HCV, Rinovirus, tick-borne viruses, Ebola, Lassa, RSV, adenoviruses, enteroviruses, metapneumoviruses, Eastern, Western, and Venezuelan Equine Encephalitis (EEE, WEE and VEE, respectively), and Chikungunya fever (CHIK), are also disclosed.
  • Coronaviridae such as MERSr-CoV, SARS-CoV-1, SARS-CoV-2, HCoV-OC43, HCoV- 229E, HCoV-
  • Figures 1A-D are charts showing the formation of 2’deoxy-NHC-DP and 2’deoxy- NHC-TP after reduction of NHC-DP by M1 ribonucleotide reductase (RNR), in terms of relative abundance (%) over time (min).
  • RNR ribonucleotide reductase
  • Non-limiting examples of suitable alkynyl groups include ethynyl, propynyl, hydroxypropynyl, butyn-l-yl, butyne- yl, pentyn-l-yl, pentyn-2-yl, 4-methoxypentyn-2-yl, 3-methylbutyn-l-yl, hexyn-l-yl, hexyn-2- yl, and hexyn-3-yl, 3,3-dimethylbutyn-l-yl radicals.
  • alkylamino or “arylamino” refers to an amino group that has one or two alkyl or aryl substituents, respectively.
  • alkoxy and “alkoxyalkyl” embrace linear or branched oxy-containing radicals having alkyl moieties, such as methoxy radical.
  • alkoxyalkyl also embraces alkyl radicals having one or more alkoxy radicals attached to the alkyl radical, that is, to form monoalkoxyalkyl and dialkoxyalkyl radicals.
  • the “alkoxy” radicals can be further substituted with one or more halo atoms, such as fluoro, chloro or bromo, to provide “haloalkoxy” radicals.
  • heteroaryl or “heteroaromatic,” as used herein, refer to an aromatic that includes at least one sulfur, oxygen, nitrogen or phosphorus in the aromatic ring.
  • the heteroaromatic group can be optionally substituted as described above for aryl.
  • the heterocyclic or heteroaromatic group can be optionally substituted with one or more substituents selected from the group consisting of halogen, haloalkyl, alkyl, alkoxy, hydroxy, carboxyl derivatives, amido, amino, alkylamino, and dialkylamino.
  • the heteroaromatic can be partially or totally hydrogenated as desired.
  • dihydropyridine can be used in place of pyridine. Functional oxygen and nitrogen groups on the heterocyclic or heteroaryl group can be protected as necessary or desired.
  • the term “host,” as used herein, refers to a unicellular or multicellular organism in which the virus can replicate, including but not limited to cell lines and animals, and, preferably, humans. Alternatively, the host can be carrying a part of the viral genome, whose replication or function can be altered by the compounds of the present invention.
  • the term host specifically refers to infected cells, cells transfected with all or part of the viral genome and animals, in particular, primates (including but not limited to chimpanzees) and humans. In most animal applications of the present invention, the host is a human being.
  • Veterinary applications in certain indications, however, are clearly contemplated by the present invention (such as for use in treating chimpanzees).
  • nucleoside also includes ribonucleosides, and representative ribonucleosides are disclosed, for example, in the Journal of Medicinal Chemistry, 43(23), 4516-4525 (2000), Antimicrobial Agents and Chemotherapy, 45(5), 1539-1546 (2001), and PCT WO 2000069876.
  • peptide refers to a natural or synthetic compound containing two to one hundred amino acids linked by the carboxyl group of one amino acid to the amino group of another.
  • pharmaceutically acceptable salt or prodrug is used throughout the specification to describe any pharmaceutically acceptable form (such as an ester) compound which, upon administration to a patient, provides the compound.
  • Pharmaceutically-acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art.
  • prodrugs refer to a compound that is metabolized, for example, hydrolyzed or oxidized, in the host to form the compound of the present invention.
  • Typical examples of prodrugs include compounds that have biologically labile protecting groups on functional moieties of the active compound.
  • Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound.
  • the prodrug forms of the compounds of this invention can possess antiviral activity, can be metabolized to form a compound that exhibits such activity, or both.
  • the compounds have the following formula: or a pharmaceutically acceptable salt or prodrug thereof, wherein: X is CH 2 , -CH(CH 3 )-, CH(CH 3 ) 2 -. CHF, CF 2 or CD 2 ,
  • Y is N or CR’
  • Z is N or CR
  • R’ is H, deuterium or fluorine
  • R is H, deuterium or methyl
  • R 1 is OH, an optionally substituted O-linked amino acid, -O-C(O)-C 1-12 alkyl, -O-C(O)- C 2-12 alkenyl, -O-C(O)-C 2-12 alkynyl, -O-C(O)-C 3-6 cycloalkyl, -O-C(O)O-C 1-12 alkyl, -O- C(O)O-C 2-12 alkenyl, -O-C(O)O-C 2-12 alkynyl, -O-C(O)O-C 3-6 cycloalkyl, OC 1-6 alkyl, OC 1-6 haloalkyl, OC 1-6 alkoxy, OC 2-6 alkenyl, OC 2-6 alkynyl, OC 3-6 cycloalkyl, O-P(O)R 6 R 7 , O-CH 2 - P-(OH) 3 , O-CH 2 -P-(OH
  • R 6 and R 7 are independently selected from the group consisting of:
  • R 15 selected from the group consisting of H, Li, Na, K, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 3-6 cycloalkyl, C 1-4 (alkyl)aryl, benzyl, C 1-6 haloalkyl, C 2-3 (alkyl)OC 1-20 alkyl, aryl, and heteroaryl, such as phenyl and pyridinyl, wherein aryl and heteroaryl are optionally substituted with zero to three substituents independently selected from the group consisting of (CH 2 ) 0-6 CO 2 R 16 and (CH 2 ) 0-6 CON(R 16 ) 2 ; where R 16 is independently H, substituted or unsubstituted C 1-20 alkyl, the carbon chain derived from a fatty alcohol or C 1-20 alkyl substituted with a C 1-6 alkyl, C 1-6 alkoxy, di(C 1-6 alkyl)-amino, flu
  • R 2 is H, deuterium, F, CN, N 3 , C 1-3 alkyl, C 2-3 alkynyl,
  • R 3 is H, deuterium, CN, N 3 , C 1-3 alkyl, C 2-3 alkynyl,
  • R 5 is H, an optionally substituted O-linked amino acid, -C(O)-C 2-12 alkyl, -C(O)-C 2-12 alkenyl, -C(O)-C 2-12 alkynyl, -C(O)-C 3-6 cycloalkyl, -C(O)O-C 2-12 alkyl, -C(O)O-C 2-12 alkenyl, -C(O)O-C 2-12 alkynyl, -C(O)O-C 3-6 cycloalkyl, wherein the groups can be substituted with one or more fluorine,
  • R 8 and R 8' are independently selected from the group consisting of H, an optionally substituted O-linked amino acid, -C(O)-C 2-12 alkyl, -C(O)-C 2-12 alkenyl, -C(O)-C 2-12 alkynyl, - C(O)-C 3-6 cycloalkyl, -C(O)O-C 2-12 alkyl, -C(O)O-C 2-12 alkenyl, -C(O)O-C 2-12 alkynyl, - C(O)O-C 3-6 cycloalkyl, wherein the groups can be substituted with one or more substituents selected from the group consisting of fluorine, hydroxyl, amino, alkylamino, arylamino, and alkoxy,
  • R 9 is H or deuterium
  • R 10 is deuterium or methyl, with the proviso that Y and Z are not both N.
  • X is -CH 2 -.
  • one or both of Y and Z are CH.
  • R 1 is O-P(O)R 6 R 7 , and R 5 and R 6 are defined such that R 1 is a phosphoramidate.
  • R 1 is OH
  • R 1 is -O-C(O)-C 2-12 alkyl.
  • R 2 is deuterium.
  • R 3 is CN or N 3 .
  • R 3 is H.
  • R 4 is 0.
  • R 5 is H.
  • one or R 6 and R 7 is the ester of a D- or L-amino acid or O-aryl, and the other is O-aryl.
  • R 8 and R 8' are H.
  • R 8 is -C(O)-C 2-12 alkyl.
  • R 9 is D.
  • R 10 is deuterium
  • R 10 is methyl
  • the compounds have the following formula: or a pharmaceutically acceptable salt or prodrug thereof, wherein:
  • X is CH 2 , -CH(CH 3 )-, CH(CH 3 ) 2 -. CHF, CF 2 or CD 2 ,
  • Y is N or CR’
  • Z is N or CR
  • R’ is H, deuterium or fluorine
  • R is H, deuterium or methyl
  • R 2 is H, deuterium, F, CN, N 3 , C 1-3 alkyl, or C 2-3 alkynyl
  • R 3 is H, deuterium, CN, N 3 , C 1-3 alkyl, or C 2-3 alkynyl
  • R 5 is H, an optionally substituted O-linked amino acid, -C(O)-C 2-12 alkyl, -C(O)-C 2-12 alkenyl, -C(O)-C 2-12 alkynyl, -C(O)-C 3-6 cycloalkyl, -C(O)O-C 2-12 alkyl, -C(O)O-C 2-12 alkenyl, -C(O)O-C 2-12 alkynyl, -C(O)O-C 3-6 cycloalkyl, wherein the groups can be substituted with one or more fluorine atoms, R 8' is selected from the group consisting of H, an optionally substituted O-linked amino acid, -C(O)-C 2-12 alkyl, -C(O)-C 2-12 alkenyl, -C(O)-C 2-12 alkynyl, -C(O)-C 3-6 cycloalkyl, - C(O
  • R 9 is H or deuterium
  • R 10 is deuterium or methyl
  • R 11 is 0 or S
  • R 12 is selected from the group consisting of:
  • R 15 is selected from the group consisting of H, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 3-6 cycloalkyl, C 1-4 (alkyl)aryl, benzyl, C 1- 6 haloalkyl, C 2-3 (alkyl)OC 1-20 alkyl, aryl, and heteroaryl, such as phenyl and pyridinyl, wherein aryl and heteroaryl are optionally substituted with zero to three substituents independently selected from the group consisting of (CH 2 ) 0-6 CO 2 R 16 and (CH 2 ) 0-6 CON(R 16 ) 2 ;
  • R 17 and R 18 are independently H, C 1-20 alkyl, the carbon chain derived from a fatty alcohol or C 1-20 alkyl optionally substituted with a C 1-6 alkyl, alkoxy, di(C 1-6 alkyl)- amino, fluoro, C 3-10 cycloalkyl, cycloalkyl-C 1-6 alkyl, cycloheteroalkyl, aryl, heteroaryl, substituted aryl, or substituted heteroaryl; wherein the substituents are C 1-5 alkyl, or C 1-5 alkyl substituted with a C 1-6 alkyl, alkoxy, di(C 1-6 alkyl)-amino, fluoro, C 3-10 cycloalkyl, or cycloalkyl; and where R 30 is selected from the group consisting of substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 3-6
  • X is -CH 2 -.
  • one or both of Y and Z are CH.
  • R 2 is deuterium
  • R 3 is CN or N 3 .
  • R 3 is H.
  • R 4 is 0.
  • R 5 is H.
  • R 8' is H.
  • R 9 is D.
  • R 10 is deuterium
  • R 10 is methyl
  • R 11 is 0.
  • R 12 is the ester of a D- or L-amino acid
  • the compounds have the following formula:
  • R 1 is OH, an optionally substituted O-linked amino acid, -O-C(O)-C 2-12 alkyl, -O-C(O)- C 2-12 alkenyl, -O-C(O)-C 2-12 alkynyl, -O-C(O)-C 3-6 cycloalkyl, -O-C(O)O-C 2-12 alkyl, -O- C(O)O-C 2-12 alkenyl, -O-C(O)O-C 2-12 alkynyl, -O-C(O)O-C 3-6 cycloalkyl, OC 1-6 alkyl, OC 1-6 haloalkyl, OC 1-6 alkoxy, OC 2-6 alkenyl, OC 2-6 alkynyl, OC 3-6 cycloalkyl, O-P(O)R 6 R 7 , O-CH 2 - P-(OH) 3 , O-CH 2 -P-(OH
  • R 6 and R 7 are independently selected from the group consisting of:
  • R 15 selected from the group consisting of H, Li, Na, K, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 3-6 cycloalkyl, C 1-4 (alkyl)aryl, benzyl, C 1-6 haloalkyl, C 2-3 (alkyl)OC 1-20 alkyl, aryl, and heteroaryl, such as phenyl and pyridinyl, wherein aryl and heteroaryl are optionally substituted with zero to three substituents independently selected from the group consisting of (CH 2 ) 0-6 CO 2 R 16 and (CH 2 ) 0-6 CON(R 16 ) 2 ; where R 16 is independently H, substituted or unsubstituted C 1-20 alkyl, the carbon chain derived from a fatty alcohol or C 1-20 alkyl substituted with a C 1-6 alkyl, C 1-6 alkoxy, di(C 1-6 alkyl)-amino, flu
  • R 17 , R 17' and R 18 are independently H, C 1-20 alkyl, the carbon chain derived from a fatty alcohol or C 1-20 alkyl optionally substituted with a C 1-6 alkyl, alkoxy, di(C 1-6 alkyl)- amino, fluoro, C 3-10 cycloalkyl, cycloalkyl-C 1-6 alkyl, cycloheteroalkyl, aryl, heteroaryl, substituted aryl, or substituted heteroaryl; wherein the substituents are C 1-5 alkyl, or C 1-5 alkyl substituted with a C 1-6 alkyl, alkoxy, di(C 1-6 alkyl)-amino, fluoro, C 3-10 cycloalkyl, or cycloalkyl;
  • R 2 is H, deuterium, F, CN, N 3 , C 1-3 alkyl, or C 2-3 alkynyl,
  • R 5 is H, an optionally substituted O-linked amino acid, -C(O)-C 2-12 alkyl, -C(O)-C 2-12 alkenyl, -C(O)-C 2-12 alkynyl, -C(O)-C 3-6 cycloalkyl, -C(O)O-C 2-12 alkyl, -C(O)O-C 2-12 alkenyl, -C(O)O-C 2-12 alkynyl, -C(O)O-C 3-6 cycloalkyl, wherein the groups can be substituted with one or more fluorine atoms,
  • R 8 and R 8' are independently selected from the group consisting of H, an optionally substituted O-linked amino acid, -C(O)-C 2-12 alkyl, -C(O)-C 2-12 alkenyl, -C(O)-C 2-12 alkynyl, - C(O)-C 3-6 cycloalkyl, -C(O)O-C 2-12 alkyl, -C(O)O-C 2-12 alkenyl, -C(O)O-C 2-12 alkynyl, - C(O)O-C 3-6 cycloalkyl, wherein the groups can be substituted with one or more substituents selected from the group consisting of fluorine, hydroxyl, amino, alkylamino, arylamino, and alkoxy,
  • R 9 is H or deuterium
  • R 10 is deuterium or methyl.
  • X is -CH 2 -.
  • one or both of Y and Z are CH.
  • R 1 is O-P(O)R 6 R 7 , and R 5 and R 6 are defined such that R 1 is a phosphoramidate.
  • R 1 is OH
  • R 1 is -O-C(O)-C 2-12 alkyl.
  • R 9 is D.
  • R 10 is methyl
  • R 9 is H or deuterium
  • R 12 is selected from the group consisting of:
  • R 17 and R 18 are independently H, C 1-20 alkyl, the carbon chain derived from a fatty alcohol or C 1-20 alkyl optionally substituted with a C 1-6 alkyl, alkoxy, di(C 1-6 alkyl)- amino, fluoro, C 3-10 cycloalkyl, cycloalkyl -C 1-6 alkyl, cycloheteroalkyl, aryl, heteroaryl, substituted aryl, or substituted heteroaryl; wherein the substituents are C 1-5 alkyl, or C 1-5 alkyl substituted with a C 1-6 alkyl, alkoxy, di(C 1-6 alkyl)-amino, fluoro, C 3-10 cycloalkyl, or cycloalkyl; and where R 30 is selected from the group consisting of substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 3-6
  • R 9 is D.
  • R 30 is -NH-OH, NH-NH 2 , -NH-OMe, -N(Me)-NH 2 , or -NH-OR 5 ,
  • R 31 is H, F, or NH 2 ,
  • R 2 is deuterium
  • R 4 is 0.
  • R 5 is H.
  • one or R 6 and R 7 is the ester of a D- or L-amino acid or O-aryl, and the other is O-aryl.
  • the compound has the formula: wherein:
  • R 5 is H.
  • R 8 is -C(O)-C 2-12 alkyl.
  • R 9 is D.
  • R 10 is deuterium
  • the compound has the following formula: wherein:
  • R 2 is deuterium
  • R 4 is 0.
  • R 11 is 0.
  • R 30 is -NH-OH, NH-NH 2 , -NH-OMe, -N(Me)-NH 2 , or -NH-OR 5 ,
  • R 31 is H, F, or NH 2
  • R 2 , R 3 , R 4 , R 5 , R 8' , R 9 , R 10 , R 11 and R 12 are as defined in Formulas A and B.
  • R 3 is CN or Ns.
  • R 3 is H.
  • R 4 is 0.
  • R 8' is H.
  • R 10 is deuterium
  • R 10 is methyl
  • R 12 is the ester of a D- or L-amino acid
  • the compound has one of the following formulas: or a pharmaceutically acceptable salt or prodrug thereof.
  • the compound has one of the following formulas:
  • any of the compounds of Formulas A-H can be present in the form of 5’-valine or isobutyl esters.
  • the disclosure relates to method of making compounds disclosed herein by mixing starting materials and reagents disclosed herein under conditions such that the compounds are formed.
  • nucleosides 4 can be prepared by first preparing nucleosides 4, which in turn can be accomplished by one of ordinary skill in the art, using methods outlined in: (a) Rajagopalan, P.; Boudinot, F. D; Chu, C. K.; Tennant, B. C.; Baldwin, B. H.; Antiviral Nucleosides: Chiral Synthesis and Chemotheraphy: Chu, C. K.; Eds. Elsevier: 2003. b) Recent Advances in Nucleosides: Chemistry and Chemotherapy: Chu, C. K.; Eds. Elsevier: 2002. c) Frontiers in Nucleosides & Nucleic Acids, 2004, Eds. R. F.
  • nucleosides 2 can be prepared by coupling sugar 1 with a protected, silylated or free nucleoside base in the presence of Lewis acid such as TMSOTf. Deprotection of the 3’- and 5’- hydroxyls gives nucleoside 3.
  • Selective protection of the 2’- and 5’- positions can be accomplished using well established sequences such as 1) reaction with 1,3 -dichloro- 1, 1,3,3-tetraisopropyldisiloxan (TIPSCI 2 ) in presence of a base such as pyridine , 2) protection of the 3 ’-hydroxyl with a Trityl group with TrCl in presence of a base, 3) selective deprotection of the 3’ and 5’ - position using , in the case of a TIPS group, Bu 4 NF salts and 4) selective protection of the 5’-position by reaction with for instance TrCl or TBDMSC1 in presence of a base.
  • TIPSCI 2 1,3 -dichloro- 1, 1,3,3-tetraisopropyldisiloxan
  • a deuterated reducing agent such as NaBD 4
  • protection of the 2’ -hydroxyl position with, for instance TBDMSC1 in presence of a base will give access to deuterated intermediate 4.
  • Deuterated nucleosides intermediates of general formula 6 can also be synthesized from deuterated intermediates by adapting the chemistry described in scheme 2 and in Ajmera et al., Labelled Compd. 1986, 23, 963; Sinhababu, et al., J. Am. Chem. Soc. 1985, 107, 7628; Robins, et al., Org. Chem. 1990, 55, 410; David, S. and Eustache, J., Carbohyd. Res. 1971, 16, 46 and David, S. and Eustache, J., Carbohyd. Res. 1971, 20.
  • Compounds of formula 9 can be prepared by adapting the chemistry descibed in Schemes 3 and 4.
  • Compound 7 can be activated by, for instance reaction with POCl 3 , or 2,4,6- triisopropylbenzenesulfonyl chloride in presence of base such as pyridine to give intermediate 8.
  • compound 9 can be prepared by reaction of nucloeside 10 with either HONH 2 -HCI or BnONH 2 -HCl followed by complete deprotection of the corresponding compound.
  • Compounds of Formulas A or B can also be prepared by adapting the chemistry described above and in: PCT WO 2019/113462.
  • Prodrugs of the desired compounds can be prepared by adapting the chemistry in: Pradere et al. Chem. Rev. 9154 (2014), or in PCT WO 2016/094677.
  • Tetrahedron 1987, 43, 2355 described the synthesis of all 2',2"-dideuterio-2'-deoxynucleosides, for both deoxy and ribonucleosides, starting with oxidation of C2' of sugar and subsequent reduction with NaBD 4 or LiAlD 4 followed by deoxygenation by tributyltin deuteride.
  • Roy et al. J. Am. Chem. Soc. 1986, 108, 1675 reported 2',2'-dideuterio-2'-deoxyguanosine and thymidine can be prepared from 2-deoxyribose 5-phosphate using 2-deoxyribose 5-phosphate aldolase enzyme in 2 H 2 O achieving some 90 atom % deuteration.
  • the synthesis of 4',5',5'- 2 H 3 -guanosine can be carried out.
  • a useful alternative method of stereospecific deuteration was developed to synthesize polydeuterated sugars.
  • This method employed exchange of hydrogen with deuterium at the hydroxyl bearing carbon (i.e. methylene and methine protons of hydroxyl bearing carbon) using deuterated Raney nickel catalyst in 2 H 2 O.
  • deuterated phenols The synthesis of deuterated phenols is described, for example, in Hoyer, H. (1950), Synthese des pan-Deutero-o-nitro-phenols. Chem. Ber., 83: 131-136. This chemistry can be adapted to prepare substituted phenols with deuterium labels. Deuterated phenols, and substituted analogs thereof, can be used, for example, to prepare phenoxy groups in phosphoramidate prodrugs.
  • deuterated amino acids The synthesis of deuterated amino acids is described, for example, in Matthews et al., Biochimica et Biophy si ca Acta (BBA) - General Subjects, Volume 497, Issue 1, 29 March 1977, Pages 1-13. These and similar techniques can be used to prepare deuterated amino acids, which can be used to prepare phosphoramidate prodrugs of the nucleosides described herein.
  • One method for synthesizing a deuterated analog of the compounds described herein involves synthesizing a deuterated ribofuranoside with a 4’-alkynyl substitution; and attaching a nucleobase to the deuterated ribofuranoside to form a deuterated nucleoside.
  • a prodrug such as a phosphorami date prodrug, can be formed by modifying the 5 ’-OH group on the nucleoside. Where a deuterated phenol and/or deuterated amino acid is used, one can prepare a deuterated phosphoramidate prodrug.
  • Another method involves synthesizing a ribofuranoside with 4’-alkynyl substitution, and attaching a deuterated nucleobase to form a deuterated nucleoside.
  • This method can optionally be performed using a deuterated furanoside to provide additional deuteration.
  • the nucleoside can be converted into a prodrug form, which prodrug form can optionally include additional deuteration.
  • a third method involves synthesizing a ribofuranoside with 4’-alkynyl substitution, attaching a nucleobase to form a nucleoside, and converting the nucleoside to a phosphoramidate prodrug using one or both of a deuterated amino acid or phenol analog in the phosphoramidate synthesis.
  • a representative synthesis for preparing the deuterated nucleosides described herein can be adapted the chemistry described in Ajmera et al., Labelled Compd. 1986, 23, 963; Sinhababu, et al., J. Am. Chem. Soc. 1985, 107, 7628; Robins, et al., Org. Chem. 1990, 55, 410; David, S. and Eustache, J., Carbohyd. Res. 1971, 16, 46 and David, S. and Eustache, J., Carbohyd. Res. 1971, 20: Accordingly, using the techniques described above, one can provide one or more deuterium atoms in the sugar, base, and/or prodrug portion of the nucleoside compounds described herein.
  • the compounds described herein can have asymmetric centers and occur as racemates, racemic mixtures, individual diastereomers or enantiomers, with all isomeric forms being included in the present invention.
  • Compounds of the present invention having a chiral center can exist in and be isolated in optically active and racemic forms. Some compounds can exhibit polymorphism.
  • the present invention encompasses racemic, optically-active, polymorphic, or stereoisomeric forms, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein.
  • optically active forms can be prepared by, for example, resolution of the racemic form by recrystallization techniques, by synthesis from optically- active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase or by enzymatic resolution.
  • One can either purify the respective compound, then derivatize the compound to form the compounds described herein, or purify the compound themselves.
  • Optically active forms of the compounds can be prepared using any method known in the art, including but not limited to by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.
  • Examples of methods to obtain optically active materials include at least the following. i) physical separation of crystals: a technique whereby macroscopic crystals of the individual enantiomers are manually separated. This technique can be used if crystals of the separate enantiomers exist, i.e., the material is a conglomerate, and the crystals are visually distinct; ii) simultaneous crystallization: a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state; iii) enzymatic resolutions: a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme; iv) enzymatic asymmetric synthesis: a synthetic techni que whereby at 1 east one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer; v) chemical a
  • first- and second-order asymmetric transformations a technique whereby diastereomers from the racemate equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer.
  • kinetic resolutions this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non- racemic reagent or catalyst under kinetic conditions; ix) enantiospecific synthesis from non-racemic precursors: a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis; x) chiral liquid chromatography: a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase (including but not limited to via chiral HPLC).
  • the stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions;
  • chiral gas chromatography a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase;
  • extraction with chiral solvents a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent;
  • xiii) transport across chiral membranes a technique whereby a racemate is placed in contact with a thin membrane barrier.
  • the barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through.
  • Chiral chromatography including but not limited to simulated moving bed chromatography, is used in one embodiment.
  • a wide variety of chiral stationary phases are commercially available.
  • pharmaceutically acceptable salts includes acid addition and base salts thereof.
  • Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form non-toxic salts, i.e., which form a physiological-acceptable anion, and
  • Suitable acid addition salts are formed from acids. Examples include the acetate, adipate, ascorbate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, a-glycerophosphate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, a-ketoglutarate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen
  • Suitable inorganic salts can also be formed, including but not limited to, sulfate, nitrate, bicarbonate and carbonate salts.
  • Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
  • Hemisalts of acids and bases can also be formed, for example, hemisulphate and hemicalcium salts.
  • suitable salts see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley -VCH, 2002), incorporated herein by reference.
  • fatty acid salts of the compounds described herein can help penetrate the stratum comeum.
  • suitable salts include salts of the compounds with stearic acid, oleic acid, lineoleic acid, palmitic acid, caprylic acid, and capric acid.
  • alkylation, acylation or other lipophilic modification of the mono, di or triphosphoate of the nucleoside will increase the stability of the nucleotide.
  • substituent groups that can replace one or more hydrogens on the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Many are described in R Jones and N. Bischofberger, Antiviral Research, 27 (1995) 1-17. Any of these can be used in combination with the disclosed nucleosides to achieve a desired effect.
  • nucleotide prodrugs are described in the following references.
  • Alky hydrogen phosphonate derivatives of the compounds described herein may be less toxic than the parent nucleoside analogues.
  • prodrug forms including 5 ’-phosphate prodrugs, can be prepared for the nucleosides described herein.
  • the disclosure relates to methods of treating or preventing a viral infection, comprising administering an effective amount of a compound or pharmaceutical composition disclosed herein to a subject in need thereof
  • the compound is administered via pulmonary administration, such as by inhalation or nebulization.
  • the compound is administered orally, topically, intraveneously, intraarticularly, intramuscularly, subcutaneously, buccally, or transdermally.
  • the viral infection is, or is caused by, an alphavirus, flavivirus, coronavirus, picomavirus, orthomyxoviridae or paramyxoviridae, RSV, influenza, Powassan virus, filoviridae or Ebola.
  • a method of treating or preventing a Zika virus infection comprising administering an effective amount of a compound or pharmaceutical composition disclosed herein to a subject in need thereof.
  • the subject is at risk of, exhibiting symptoms of, or diagnosed with an infection by an RNA virus, such as an influenza A virus, such as subtype H1N1, H3N2, H7N9, or H5N1, influenza B virus, influenza C virus, rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, human coronavirus, such as MERSr-CoV, SARS-CoV-1, SARS-CoV- 2, HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1, human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, parvovirus B 19, molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cell polyomavirus, coxsackie A virus, norovirus
  • influenza A virus including subtypes H1N1, H3N2, H7N9, H5N1 (low path), and H5N1 (high path) influenza B virus, influenza C virus, rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, SAKS coronavirus, such as MERSr-CoV, SARS-CoV-1, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1, human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, parvovirus B19, molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cell polyomavirus, coxsackie A virus, norovirus, Rubella virus, lymphoc
  • the methods described herein can be used to treat, prevent, manage or lessen the severity of symptoms and infections associated with one or more pulmonary diseases or infections in a subject.
  • the methods involve administering one or more of the compounds described herein to the subject.
  • the compounds can be administered to the mouth, nasal passages, throat, esophagus, larynx, pharynx, trachea, bronchioles, bronchi, upper airways, lower airways, subcutaneously or via an implant (for example, up under the ribs and into the chest cavity), and combinations thereof.
  • the compounds are administered during lung lavage, which can be whole lung lavage or bronchoalveolar lavage (BAL).
  • BAL also known as bronchoalveolar washing
  • a bronchoscope is passed through the mouth or nose into the lungs and fluid is squirted into a small part of the lung and then collected for examination.
  • the compounds can travel through the fluid, and treat the entire fluid-coated portion of the lung.
  • Bronchoalveolar lavage is commonly used to diagnose infections in people with immune system problems, pneumonia in people on ventilators, some types of lung cancer, and scarring of the lung (interstitial lung disease). It is the most common method used to sample the epithelial lining fluid (ELF) and to determine the protein composition of the pulmonary airways. It is often used in immunological research as a means of sampling cells (for example, T cells) or pathogen levels (for example, influenza virus) in the lung. During the procedure, the compounds described herein can be administered. Whole lung lavage (WLL; or "lung washing") is a treatment for pulmonary alveolar proteinosis. While the lung is washed, therapy with the compounds can also be administered, and the fluid allows the compounds to contact the entire fluid-coated surface of the lung.
  • WLL Whole lung lavage
  • the compounds are used to treat or prevent microbial infections, including those caused by viruses such as Coronavirus, including MERSr-CoV, SARS-CoV-1, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1.
  • viruses such as Coronavirus, including MERSr-CoV, SARS-CoV-1, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1.
  • Hosts including but not limited to humans, infected with a Coronviridae virus, or the other viruses described, herein can be treated by administering to the patient an effective amount of the active compound or a pharmaceutically acceptable prodrug or salt thereof in the presence of a pharmaceutically acceptable carrier or diluent.
  • the active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.
  • a preferred dose of the compound for will be in the range of between about 0.01 and about 10 mg/kg, more generally, between about 0.1 and 5 mg/kg, and, preferably, between about 0.5 and about 2 mg/kg, of body weight of the recipient per day, until the patient has recovered.
  • a compound may be administered at a dosage of up to 10 ⁇ M, which might be considered a relatively high dose if administered for an extended period of time, but which can be acceptable when administered for the duration of an infection with one or more of the viruses described herein, which is typically on the order of several days to several weeks.
  • the effective dosage range of the pharmaceutically acceptable salts and prodrugs can be calculated based on the weight of the parent compound to be delivered. If the salt or prodrug exhibits activity in itself, the effective dosage can be estimated as above using the weight of the salt or prodrug, or by other means known to those skilled in the art.
  • the compound is conveniently administered in unit any suitable dosage form, including but not limited to but not limited to one containing 7 to 600 mg, preferably 70 to 600 mg of active ingredient per unit dosage form.
  • An oral dosage of 5-400 mg is usually convenient.
  • the concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • the active ingredient can be administered at once, or can be divided into a number of smaller doses to be administered at varying intervals of time.
  • Oral compositions will generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or com starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compound can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
  • a symp can contain, in addition to the active compound(s), sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the compound or a pharmaceutically acceptable prodmg or salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, anti- inflammatories or other antiviral compounds.
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for the adjustment of tonicity, such as sodium chloride or dextrose.
  • the parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • preferred carriers are physiological saline or phosphate buffered saline (PBS).
  • the compositions are present in the form of transdermal formulations, such as that used in the FDA-approved agonist rotigitine transdermal (Neupro patch).
  • Another suitable formulation is that described in U.S. Publication No. 20080050424, entitled “Transdermal Therapeutic System for Treating Parkinsonism.”
  • This formulation includes a silicone or acrylate-based adhesive, and can include an additive having increased solubility for the active substance, in an amount effective to increase dissolving capacity of the matrix for the active substance.
  • the transdermal formulations can be single-phase matrices that include a backing layer, an active substance-containing self-adhesive matrix, and a protective film to be removed prior to use. More complicated embodiments contain multiple-layer matrices that may also contain non-adhesive layers and control membranes. If a polyacrylate adhesive is used, it can be crosslinked with multivalent metal ions such as zinc, calcium, aluminum, or titanium ions, such as aluminum acetyl acetonate and titanium acetylacetonate.
  • multivalent metal ions such as zinc, calcium, aluminum, or titanium ions, such as aluminum acetyl acetonate and titanium acetylacetonate.
  • silicone adhesives When silicone adhesives are used, they are typically polydimethylsiloxanes. However, other organic residues such as, for example, ethyl groups or phenyl groups may in principle be present instead of the methyl groups. Because the active compounds are amines, it may be advantageous to use amine-resistant adhesives. Representative amine- resistant adhesives are described, for example, in EP 0 180377.
  • acrylate-based polymer adhesives include acrylic acid, acrylamide, hexyl acrylate, 2-ethylhexylacrylate, hydroxyethyl acrylate, octylacrylate, butylacrylate, methylacrylate, glycidylacrylate, methacrylic acid, methacrylamide, hexylmethacrylate, 2- ethylhexylmethacrylate, octylmethacrylate, methylmethacrylate, glycidylmethacrylate, vinylacetate, vinylpyrrolidone, and combinations thereof.
  • the adhesive must have a suitable dissolving capacity for the active substance, and the active substance most be able to move within the matrix, and be able to cross through the contact surface to the skin.
  • Those of skill in the art can readily formulate a transdermal formulation with appropriate transdermal transport of the active substance.
  • Certain pharmaceutically acceptable salts tend to be more preferred for use in transdermal formulations, because they can help the active substance pass the barrier of the stratum comeum.
  • fatty acid salts such as stearic acid and oleic acid salts.
  • Oleate and stearate salts are relatively lipophilic, and can even act as a permeation enhancer in the skin.
  • Permeation enhancers can also be used.
  • Representative permeation enhancers include fatty alcohols, fatty acids, fatty acid esters, fatty acid amides, glycerol or its fatty acid esters, N-methylpyrrolidone, terpenes such as limonene, alpha-pinene, alpha- terpineol, carvone, carveol, limonene oxide, pinene oxide, and 1,8-eucalyptol.
  • the patches can generally be prepared by dissolving or suspending the active agent in ethanol or in another suitable organic solvent, then adding the adhesive solution with stirring. Additional auxiliary substances can be added either to the adhesive solution, the active substance solution or to the active substance-containing adhesive solution. The solution can then be coated onto a suitable sheet, the solvents removed, a backing layer laminated onto the matrix layer, and patches punched out of the total laminate.
  • the compounds are administered to the pulmonary tract (i.e., via pulmonary administration), for example, via intranasal administration or nebulization.
  • pulmonary administration comprises inhalation of the compounds, typically in the form of particles or droplets, such as by nasal, oral inhalation, or both.
  • the formulations can be developed to be aerosolized via a metered dose inhaler, a dry powder inhaler, a liquid spray or a nebulizer devises. Nebulization can be accomplished by compressed air, ultrasonic energy, or vibrating mesh to form a plurality of liquid droplets or solid particles comprising the NO-releasing compounds.
  • particles may be formulated as an aerosol (i.e., liquid droplets of a stable dispersion or suspension of particles that include one or more of the compounds described herein in a gaseous medium). Particles delivered by aerosol may be deposited in the airways by gravitational sedimentation, inertial impaction, and/or diffusion.
  • aerosol i.e., liquid droplets of a stable dispersion or suspension of particles that include one or more of the compounds described herein in a gaseous medium.
  • the particles or droplets can be administered in two or more separate administrations (doses).
  • Biodegradable particles can be used for the controlled-release and delivery of the compounds described herein. Aerosols for the delivery of therapeutic agents to the respiratory tract have been developed. Adjei, A. and Garren, J. Pharm Res. 7, 565-569 (1990); and Zanen, P. and Lamm, J.-W. J. Int. J. Pharm. 114, 111-115 (1995).
  • the respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli.
  • the upper and lower airways are called the conducting airways.
  • the terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung. Gonda, I. "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract," in Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313, 1990.
  • the deep lung, or alveoli are the primary target of inhaled therapeutic aerosols for systemic drug delivery.
  • Relatively large particles tend to get trapped in the oropharyngeal cavity, which can lead to excessive loss of the inhaled drug.
  • Relatively smaller particles can be delivered to the deep lung, but can be phagocytosed.
  • One way to deliver relatively large particles (sized to avoid phagocytosis), which are light enough to avoid excessive entrapment in the oropharyngeal cavity, is to use porous particles.
  • the particles for delivering the compounds described herein to the alveolar regions of the lung are porous, “aerodynamically-light” particles, as described in U.S. Patent No. 6,977,087.
  • Aerodynamically light particles can be made of a biodegradable material, and typically have a tap density less than 0.4 g/cm 3 and a mass mean diameter between 5 pm and 30 pm.
  • the particles may be formed of biodegradable materials such as biodegradable polymers.
  • the particles may be formed of a functionalized polyester graft copolymer consisting of a linear alpha-hydroxy-acid polyester backbone having at least one amino acid group incorporated herein and at least one poly(amino acid) side chain extending from an amino acid group in the polyester backbone.
  • aerodynamically light particles having a large mean diameter, for example greater than 5 pm can be used for enhanced delivery of one or more of the compounds described herein to the alveolar region of the lung.
  • Aqueous Solutions Compounds can be administered intranasally, as well as topically, intranasally, intraveneously, by injection, and by nebulization, in aqueous solutions.
  • the solutions comprise one or more salts and are isotonic.
  • the disclosure contemplates a pressurized or unpressurized container comprising a compound or pharmaceutical composition as described herein.
  • the container is a manual pump spray, inhaler, meter-dosed inhaler, dry powder inhaler, nebulizer, vibrating mesh nebulizer, jet nebulizer, or ultrasonic wave nebulizer.
  • a composition for inhalation comprises a compound disclosed herein and a propellant.
  • the propellant is an aerosolizing propellant such as compressed air, ethanol, nitrogen, carbon dioxide, nitrous oxide, hydrofluoroalkanes (HFAs), 1,1, 1,2, -tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoropropane or combinations thereof.
  • controlled release nanoparticulate formulations comprise a nanoparticulate active agent to be administered and a rate-controlling polymer which prolongs the release of the agent following administration, hi this embodiment, the compositions can release the active agent, following administration, for a time period ranging from about 2 to about 24 hours or up to 30 days or longer.
  • Representative controlled release formulations including a nanoparticulate form of the active agent are described, for example, in U.S. Patent No. 8,293,277.
  • Nanoparticulate compositions can comprise particles of the active agents described herein, having a non-crosslinked surface stabilizer adsorbed onto, or associated with, their surface.
  • the average particle size of the nanoparticulates is typically less than about 800 nm, more typically less than about 600 nm, still more typically less than about 400 nm, less than about 300 nm, less than about 250 nm, less than about 100 nm, or less than about 50 nm. In one aspect of this embodiment, at least 50% of the particles of active agent have an average particle size of less than about 800, 600, 400, 300, 250, 100, or 50 nm, respectively, when measured by light scattering techniques.
  • a variety of surface stabilizers are typically used with nanoparticulate compositions to prevent the particles from clumping or aggregating.
  • Representative surface stabilizers are selected from the group consisting of gelatin, lecithin, dextran, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodium dodecyl sulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl- cellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanol
  • Lysozymes can also be used as surface stabilizers for nanoparticulate compositions.
  • Certain nanoparticles such as poly(lactic-co-glycolic acid) (PLGA)-nanoparticles are known to target the liver when given by intravenous (IV) or subcutaneously (SQ).
  • IV intravenous
  • SQ subcutaneously
  • Representative rate controlling polymers into which the nanoparticles can be formulated include chitosan, polyethylene oxide (PEO), polyvinyl acetate phthalate, gum arabic, agar, guar gum, cereal gums, dextran, casein, gelatin, pectin, carrageenan, waxes, shellac, hydrogenated vegetable oils, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcelluose (HPMC), sodium carboxymethylcellulose (CMC), poly(ethylene) oxide, alkyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydrophilic cellulose derivatives, polyethylene glycol, polyvinylpyrrolidone, cellulose acetate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose acetate trimellitate, polyvinyl acetate phthalate, hydroxypropylmethyl
  • Nanoparticulate compositions are also described, for example, in U.S. Pat. No. 5,298,262 for "Use of Ionic Cloud Point Modifiers to Prevent Particle Aggregation During Sterilization;” U.S. Pat. No. 5,302,401 for “Method to Reduce Particle Size Growth During Lyophilization;” U.S. Pat. No. 5,318,767 for "X-Ray Contrast Compositions Useful in Medical Imaging;” U.S. Pat. No. 5,326,552 for "Novel Formulation For Nanoparticulate X-Ray Blood Pool Contrast Agents Using High Molecular Weight Non-ionic Surfactants;" U.S. Pat. No.
  • Amorphous small particle compositions are described, for example, in U.S. Pat. No. 4,783,484 for "Particulate Composition and Use Thereof as Antimicrobial Agent;” U.S. Pat. No. 4,826,689 for “Method for Making Uniformly Sized Particles from Water- Insoluble Organic Compounds;” U.S. Pat. No. 4,997,454 for “Method for Making Uniformly- Sized Particles From Insoluble Compounds;” U.S. Pat. No. 5,741,522 for "Ultrasmall, Non- aggregated Porous Particles of Uniform Size for Entrapping Gas Bubbles Within and Methods; " and U.S. Pat. No. 5,776,496, for "Ultrasmall Porous Particles for Enhancing Ultrasound Back
  • Certain nanoformulations can enhance the absorption of drugs by releasing drug into the lumen in a controlled manner, thus reducing solubility issues.
  • the intestinal wall is designed to absorb nutrients and to act as a barrier to pathogens and macromolecules. Small amphipathic and lipophilic molecules can be absorbed by partitioning into the lipid bilayers and crossing the intestinal epithelial cells by passive diffusion, while nanoformulation absorption may be more complicated because of the intrinsic nature of the intestinal wall.
  • the first physical obstacle to nanoparticle oral absorption is the mucus barrier which covers the luminal surface of the intestine and colon.
  • the mucus barrier contains distinct layers and is composed mainly of heavily glycosylated proteins called mucins, which have the potential to block the absorption of certain nanoformulations.
  • nanoformulations across intestinal epithelial cells can be regulated by several steps, including cell surface binding, endocytosis, intracellular trafficking and exocytosis, resulting in transcytosis (transport across the interior of a cell) with the potential involvement of multiple subcellular structures.
  • nanoformulations can also travel between cells through opened tight junctions, defined as paracytosis.
  • Non-phagocytic pathways, which involve clathrin-mediated and caveolae-mediated endocytosis and macropinocytosis, are the most common mechanisms of nanoformulation absorption by the oral route.
  • Non-oral administration can provide various benefits, such as direct targeting to the desired site of action and an extended period of drug action.
  • Transdermal administration has been optimized for nanoformulations, such as solid lipid nanoparticles (SLNs) and NEs, which are characterized by good biocompatibility, lower cytotoxicity and desirable drug release modulation (Cappel and Kreuter, “Effect of nanoparticles on transdermal drug delivery. J Microencapsul 8: 369-374 (1991)).
  • Nasal administration of nanoformulations allows them to penetrate the nasal mucosal membrane, via a transmucosal route by endocytosis or via a carrier- or receptor-mediated transport process (Hlum, “Nanoparticulate systems for nasal delivery of drugs: a real improvement over simple systems?” J. Pharm. Sci 96: 473-483 (2007)), an example of which is the nasal administration of chitosan nanoparticles of tizanidine to increase brain penetration and drug efficacy in mice (Patel et al., “Improved transnasal transport and brain uptake of tizanidine HCl-loaded thiolated chitosan nanoparticles for alleviation of pain,” J. Pharm.
  • Pulmonary administration provides a large surface area and relative ease of access.
  • the mucus barrier, metabolic enzymes in the tracheobronchial region and macrophages in the alveoli are typically the main barriers for drug penetration.
  • Particle size is a major factor determining the diffusion of nanoformulation in the bronchial tree, with particles in the nano-sized region more likely to reach the alveolar region and particles with diameters between 1 and 5 pm expected to deposit in the bronchioles (Musante et al., “Factors affecting the deposition of inhaled porous drug particles,” J Pharm Sci 91: 1590-1600 (2002)).
  • Certain nanoformulations have a minimal penetration through biological membranes in sites of absorption and for these, i.v. administration can be the preferred route to obtain an efficient distribution in the body (Wacker, “Nanocarriers for intravenous injection-The long hard road to the market,” Int. J. Pharm., 457: 50-62., 2013).
  • nanoformulations can vary widely depending on the delivery system used, the characteristics of the nanoformulation, the variability between individuals, and the rate of drug loss from the nanoformulations.
  • Certain nanoparticles such as solid drug nanoparticles (SDNs), improve drug absorption, which does not require them to arrive intact in the systemic circulation. Other nanoparticles survive the absorption process, thus altering the distribution and clearance of the contained drug.
  • SDNs solid drug nanoparticles
  • Nanoformulations of a certain size and composition can diffuse in tissues through well- characterized processes, such as the enhanced permeability and retention effect, whereas others accumulate in specific cell populations, which allows one to target specific organs.
  • Complex biological barriers can protect organs from exogenous compounds, and the blood-brain barrier (BBB) represents an obstacle for many therapeutic agents.
  • BBB blood-brain barrier
  • Many different types of cells including endothelial cells, microglia, pericytes and astrocytes are present in the BBB, which exhibits extremely restrictive tight junctions, along with highly active efflux mechanisms, limiting the permeation of most drugs. Transport through the BBB is typically restricted to small lipophilic molecules and nutrients that are carried by specific transporters.
  • One of the most important mechanisms regulating diffusion of nanoformulations into the brain is endocytosis by brain capillary endothelial cells.
  • Macrophages in the liver are a major pool of the total number of macrophages in the body.
  • Kupffer cells in the liver possess numerous receptors for selective phagocytosis of opsonized particles (receptors for complement proteins and for the fragment crystallizable part of IgG).
  • Phagocytosis can provide a mechanism for targeting the macrophages, and providing local delivery (i.e., delivery inside the macrophages) of the compounds described herein (TRUE?).
  • Nanoparticles linked to polyethylene glycol (PEG) have minimal interactions with receptors, which inhibits phagocytosis by the mononuclear phagocytic system (Bazile et al., “Stealth Me.PEG-PLA nanoparticles avoid uptake by the mononuclear phagocytes system,” J. Pharm. Sci. 84: 493-498 (1995)).
  • PEG polyethylene glycol
  • Representative nanoformulations include inorganic nanoparticles, SDNs, SLNs, NEs, liposomes, polymeric nanoparticles and dendrimers.
  • the compounds described herein can be contained inside a nanoformulation, or, as is sometimes the case with inorganic nanoparticles and dendrimers, attached to the surface.
  • Hybrid nanoformulations which contain elements of more than one nanoformulation class, can also be used.
  • SDNs are lipid-free nanoparticles, which can improve the oral bioavailability and exposure of poorly water-soluble dmgs (Chan, “Nanodrug particles and nanoformulations for drug delivery,” Adv. Drug. Deliv. Rev. 63: 405 (2011)).
  • SDNs include a drug and a stabilizer, and are produced using ‘top-down’ (high pressure homogenization and wet milling) or bottom- up (solvent evaporation and precipitation) approaches.
  • SLNs consist of a lipid (or lipids) which is solid at room temperature, an emulsifier and water. Lipids utilized include, but are not limited to, triglycerides, partial glycerides, fatty acids, steroids and waxes. SLNs are most suited for delivering highly lipophilic drugs.
  • NEs Liquid droplets of less than a 1000 nm dispersed in an immiscible liquid are classified as NEs.
  • NEs are used as carriers for both hydrophobic and hydrophilic agents, and can be administered orally, transdermally, intravenously, intranasally, and ocularly. Oral administration can be preferred for chronic therapy, and NEs can effectively enhance oral bioavailability of small molecules, peptides and proteins.
  • Polymeric nanoparticles are solid particles typically around 200-800 nm in size, which can include synthetic and/or natural polymers, and can optionally be pegylated to minimize phagocytosis.
  • Polymeric nanoparticles can increase the bioavailability of drugs and other substances, compared with traditional formulations. Their clearance depends on several factors, including the choice of polymers (including polymer size, polymer charge and targeting ligands), with positively charged nanoparticles larger than 100 nm being eliminated predominantly via the liver (Alexis et al., Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm 5: 505-515 (2008)).
  • Dendrimers are tree-like, nanostructured polymers which are commonly 10-20 nm in diameter.
  • Liposomes are spherical vesicles which include a phospholipid bilayer. A variety of lipids can be utilized, allowing for a degree of control in degradation level. In addition to oral dosing, liposomes can be administered in many ways, including intravenously (McCaskill et al., 2013), transdermally (Pierre and Dos Santos Miranda Costa, 2011), intravitreally (Honda et al., 2013) and through the lung (Chattopadhyay, 2013). Liposomes can be combined with synthetic polymers to form lipid-polymer hybrid nanoparticles, extending their ability to target specific sites in the body.
  • the clearance rate of liposome-encased drugs is determined by both drug release and destruction of liposomes (uptake of liposomes by phagocyte immune cells, aggregation, pH-sensitive breakdown, etc.) (Ishida et al., “Liposome clearance,” Biosci Rep 22: 197-224 (2002)).
  • One of more of these nanoparticulate formulations can be used to deliver the active agents described herein to the macrophages, across the blood brain barrier, and other locations as appropriate.
  • the compounds described herein can be administered in combination or alternation with additional antiviral compounds.
  • anti-inflammatory compounds can be administered, and where a secondary infection is present, or is to be prevented, an antibiotic can be administered.
  • Additional types of compounds can also be administered, as discussed below, depending on the type of physiological damage the vims may cause, and/or the cytokine storm caused by the immune system may cause.
  • Representative additional active compounds include, but are not limited to, analgesics, anti-inflammatory drugs, antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine drugs, antimuscarinics, anxioltyics, sedatives, hypnotics, antipsychotics, bronchodilators, anti-asthma dmgs, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastro-intestinal dmgs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, stimulants, anorectics, anti-narcoleptics, and antiviral agents.
  • analgesics include, but are not limited to, analgesics, anti-inflammatory drugs, antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine drugs, antimuscarinics, anxioltyics, sedatives, hyp
  • the antiviral agent is a non-CNS targeting antiviral compound.
  • “Adjunctive administration”, as used herein, means the compound can be administered in the same dosage form or in separate dosage forms with one or more other active agents.
  • the additional active agent(s) can be formulated for immediate release, controlled release, or combinations thereof.
  • compounds that can be adjunctively administered with the compounds include, but are not limited to, aceclofenac, acetaminophen, adomexetine, almotriptan, alprazolam, amantadine, amcinonide, aminocyclopropane, amitriptyline, amolodipine, amoxapine, amphetamine, aripiprazole, aspirin, atomoxetine, azasetron, azatadine, beclomethasone, benactyzine, benoxaprofen, bermoprofen, betamethasone, bicifadine, bromocriptine, budesonide, buprenorphine, bupropion, buspirone, butorphanol, butriptyline, caffeine, carbamazepine, carbidopa, carisoprodol, celecoxib, chlordiazepoxide, chlorpromazine, choline salicy
  • the exemplary compounds and pharmaceutical compositions can be administered in combination with another antiviral agent(s) such as abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, balapiravir, BCX4430, boceprevir, cidofovir, combivir, daclatasvir, darunavir, dasabuvir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, favipiravir, fomivirsen, fosamprenavir, foscamet, fosfonet, ganciclovir, GS-5734, ibacitabine, imunovir, idoxuridine, imiquimod, indina
  • the compounds described herein can be combined with additional compounds useful for treating the disease states also treated by the release of NO.
  • the compounds discussed below can be used in combination therapy to treat Covid-19 infections, or other respiratory infections with similar pathology.
  • the at least one other active agent is selected from the group consisting of fusion inhibitors, entry inhibitors, protease inhibitors, polymerase inhibitors, antiviral nucleosides, such as remdesivir, GS-441524, N4-hydroxy cytidine, and other compounds disclosed in U.S. Patent No. 9,809,616, and their prodrugs, viral entry inhibitors, viral maturation inhibitors, JAK inhibitors, angiotensin-converting enzyme 2 (ACE2) inhibitors, SARS-CoV-specific human monoclonal antibodies, including CR3022, and agents of distinct or unknown mechanism.
  • fusion inhibitors entry inhibitors, protease inhibitors, polymerase inhibitors, antiviral nucleosides, such as remdesivir, GS-441524, N4-hydroxy cytidine, and other compounds disclosed in U.S. Patent No. 9,809,616, and their prodrugs, viral entry inhibitors, viral maturation inhibitors, JAK inhibitors, an
  • Umifenovir (also known as Arbidol) is a representative fusion inhibitor.
  • Representative entry inhibitors include Camostat, luteolin, MDL28170, SSAA09E2, SSAA09E1 (which acts as a cathepsin L inhibitor), SSAA09E3, and tetra-O-galloyl- ⁇ -D- glucose (TGG).
  • the chemical formulae of certain of these compounds are provided below:
  • Remdesivir, Sofosbuvir, ribavirin, IDX-184 and GS-441524 have the following formulas:
  • one can administer compounds which inhibit the cytokine storm such as dexamethasone, anti -coagulants and/or platelet aggregation inhibitors that address blood clots, or compounds which chelate iron ions released from hemoglobin by viruses such as COVID- 19.
  • compounds which inhibit the cytokine storm such as dexamethasone, anti -coagulants and/or platelet aggregation inhibitors that address blood clots, or compounds which chelate iron ions released from hemoglobin by viruses such as COVID- 19.
  • Representative ACE-2 inhibitors include sulfhydryl-containing agents, such as alacepril, captopril (capoten), and zefnopril, dicarboxylate-containing agents, such as enalapril (vasotec), ramipril (altace), quinapril (accupril), perindopril (coversyl), lisinopril (listril), benazepril (lotensin), imidapril (tanatril), trandolapril (mavik), and cilazapril (inhibace), and phosphonate-containing agents, such as fosinopril (fositen/monopril).
  • sulfhydryl-containing agents such as alacepril, captopril (capoten), and zefnopril
  • dicarboxylate-containing agents such as enalapril (vasotec), ramipril (alt
  • the active compound or its prodrug or pharmaceutically acceptable salt when used to treat or prevent infection, can be administered in combination or alternation with another antiviral agent including, but not limited to, those of the formulae above.
  • another antiviral agent including, but not limited to, those of the formulae above.
  • effective dosages of two or more agents are administered together, whereas during alternation therapy, an effective dosage of each agent is administered serially.
  • the dosage will depend on absorption, inactivation and excretion rates of the drug, as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
  • antiviral agents that can be used in combination with the compounds disclosed herein include those listed below.
  • cytokine storm a damaging systemic inflammation
  • cytokine storm A number of cytokines with anti-inflammatory properties are responsible for this, such as IL- 10 and transforming growth factor ⁇ (TGF- ⁇ ).
  • TGF- ⁇ transforming growth factor ⁇
  • Each cytokine acts on a different part of the inflammatory response. For example, products of the Th2 immune response suppress the Th1 immune response and vice versa.
  • one or more compounds which inhibit the cytokine storm can be co-administered.
  • Compounds which inhibit the cytokine storm include compounds that target fundamental immune pathways, such as the chemokine network and the cholinergic anti- inflammatory pathway.
  • JAK inhibitors such as JAK 1 and JAK 2 inhibitors
  • JAK 1 and JAK 2 inhibitors can inhibit the cytokine storm, and in some cases, are also antiviral.
  • Representative JAK inhibitors include those disclosed in U.S. Patent No. 10,022,378, such as Jakafi, Tofacitinib, and Baricitinib, as well as LY3009104/INCB28050, Pacritinib/SB1518, VX-509, GLPG0634, INC424, R-348, CYT387, TG 10138, AEG 3482, and pharmaceutically acceptable salts and prodrugs thereof.
  • HMGB1 antibodies and COX-2 inhibitors can be used, which downregulate the cytokine storm.
  • Examples of such compounds include Actemra (Roche).
  • Celebrex (celecoxib), a COX-2 inhibitor, can be used.
  • IL-8 (CXCL8) inhibitors can also be used.
  • Chemokine receptor CCR2 antagonists such as PF-04178903 can reduce pulmonary immune pathology.
  • Selective a7Ach receptor agonists such as GTS-21 (DMXB-A) and CNI-1495, can be used. These compounds reduce TNF-a.
  • Compounds for Treating or Preventing Blood Clots Viruses that cause respiratory infections can be associated with pulmonary blood clots, and blood clots that can also do damage to the heart.
  • Integrilin® is typically administered at a dosage of 180 mcg/kg intravenous bolus administered as soon as possible following diagnosis, with 2 mcg/kg/min continuous infusion (following the initial bolus) for up to 96 hours of therapy.
  • Representative anti-coagulants include coumarins (vitamin K antagonists), heparin and derivatives thereof, including unfractionated heparin (UFH), low molecular weight heparin (LMWH), and ultra-low-molecular weight heparin (ULMWH), synthetic pentasaccharide inhibitors of factor Xa, including Fondaparinux, Idraparinux, and Idrabiotaparinux, directly acting oral anticoagulants (DAOCs), such as dabigatran, rivaroxaban, apixaban, edoxaban and betrixaban, and antithrombin protein therapeutics/thrombin inhibitors, such as bivalent drugs hirudin, lepirudin, and bivalirudin and monovalent argatroban.
  • DAOCs directly acting oral anticoagulants
  • antithrombin protein therapeutics/thrombin inhibitors such as bivalent drugs hirudin, lepirudin, and bivalirudin and monovalent argatroban.
  • Representative platelet aggregation inhibitors include pravastatin, Plavix (clopidogrel bisulfate), Pletal (cilostazol), Effient (prasugrel), Aggrenox (aspirin and dipyridamole), Brilinta (ticagrelor), caplacizumab, Kengreal (cangrelor), Persantine (dipyridamole), Ticlid (ticlopidine), Yosprala (aspirin and omeprazole).
  • pravastatin Plavix (clopidogrel bisulfate), Pletal (cilostazol), Effient (prasugrel), Aggrenox (aspirin and dipyridamole), Brilinta (ticagrelor), caplacizumab, Kengreal (cangrelor), Persantine (dipyridamole), Ticlid (ticlopidine), Yosprala (aspirin and omeprazole).
  • Additional Compounds that can be used in combination therapy include the following: Antibodies, including monoclonal antibodies (mAb), Arbidol (umifenovir), Actemra (tocilizumab), APN01 (Aperion Biologies), ARMS-1 (which includes Cetylpyridinium chloride (CPC)), ASC09 (Ascletis Pharma), AT-001 (Applied Therapeutics Inc.) and other aldose reductase inhibitors (ARI), ATYR1923 (aTyr Pharma, Inc.), Aviptadil (Relief Therapeutics), Azvudine, Bemcentinib, BLD-2660 (Blade Therapeutics), Bevacizumab, Brensocatib, Calquence (acalabrutinib), Camostat mesylate (a TMPRSS2 inhibitor), Camrelizumab, CAP- 1002 (Capri cor Therapeutics), CD24Fcm, Clevudine, (On), mAb), Arbido
  • Therapeutic agents include, but are not limited to, chemotherapeutic agents, such as doxorubicin; dexamethasone; anti-infective agents, such as antibiotics (e.g. tetracycline, streptomycin, amphotericin and isoniazid), heavy metals such as antimony (e.g.
  • pentavalent antimonials such as DNA, RNA, RNAi, siRNA, CpG or Poly (I:C); peptides; proteins; or metals such as silver, gallium or gadolinium, paromomycin, miltefosine, fluconazole, pentamide, Meglumine antimoniate, and combinations thereof.
  • the additional therapeutic agent is an antimicrobial drug selected from the group comprising: an antibiotic; an anti-tuberculosis antibiotic (such as isoniazid, streptamycin, or ethambutol); drugs with effect on Zika virus; drugs with effect on Dengue virus, drugs with effect on Yellow Fever, and drugs with effect on family Flaviviridae viruses.
  • an antibiotic such as isoniazid, streptamycin, or ethambutol
  • drugs with effect on Zika virus drugs with effect on Dengue virus, drugs with effect on Yellow Fever, and drugs with effect on family Flaviviridae viruses.
  • the additional therapeutic agent is selected from the group consisting of cytostatic agents, alkylating agents, antimetabolites, anti-proliferative agents, tubulin binding agents, hormones and hormone antagonists, anthracycline drugs, vinca drugs, mitomycins, bleomycins, cytotoxic nucleosides, pteridine drugs, diynenes, podophyllotoxins, toxic enzymes, and radiosensitizing drugs.
  • Representative therapeutic agents include mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, triaziquone, nitrosourea compounds, adriamycin, carminomycin, daunorubicin (daunomycin), doxorubicin, isoniazid, indomethacin, gallium(III),
  • therapeutic agent is a hormone or hormone antagonist
  • representative additional therapeutic agents include prednisone, hydroxyprogesterone, medroprogesterone, diethylstilbestrol, tamoxifen, testosterone, and aminogluthetimide and combinations thereof.
  • Additional compounds that can be co-administered include phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate containing prodrugs, peptide containing prodrugs, (-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosinem, and 5-fluorouridine prodrugs that can be converted to the more active cytotoxic free drug and combinations thereof.
  • retinoic acid analogues such as those disclosed in U.S. Patent No. 11,007,160.
  • Retinoids are a class of natural and synthetic vitamin A-derivatives in which the terminal carboxyl group of retinoic acid is linked to an aminophenol residue.
  • Representative retinoic acid analogues include N-(4-hydroxyphenyl) retinamide (4-HPR), also known as Fenretinide, and 4-HPR metabolites, such as N-(4- hydroxyphenyl)-4-oxoretinamide (4-oxo-4-HPR).
  • NSAIDS and other suitable pain relievers can also be used to help treat the associated symptoms of certain Flaviridae viruses, including DENV, YFV, WNV, or JEV infection.
  • Celgosivir and analogs of celgosivir disclosed in U.S. Patent No. 11,000,516 can also be co-administered.
  • the compounds described herein can also be administered in combination with a protease inhibitor, NS5 A inhibitor, polymerase inhibitor, ribavirin, interferon, an RNA helicase DDX3 inhibitor, such as those disclosed in U.S. Patent No. 10,941,121, a mono- or di- substituted indole or substituted indolene Dengue viral replication inhibitors, such as those disclosed in U.S. Patent Nos.
  • an adaptor-associated kinase 1 (AAK1) inhibitor or cyclin G-associated kinase (GAK) inhibitor such as sunitinib, erlotinib, PKC-412 or midostaurin, toll-like receptor (TLR) agonists, or P-selectin glycoprotein ligand- 1 (PSGL-1) agonists or antagonists.
  • the deuterated compounds described herein are combined with protease inhibitors or other non-C or U nucleoside antiviral analogs.
  • protease inhibitors or other non-C or U nucleoside antiviral analogs are combined with protease inhibitors or other non-C or U nucleoside antiviral analogs.
  • N 4 -hydroxycytidine is known to be mutagenic (see, for example, Celina Janion, Barry W. Glickman, “N4-hydroxycytidine: A mutagen specific for at to GC transitions, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis,” Volume 72, Issue 1, pages 43-47 (1980)).
  • N 4 -hydroxycytidine can be converted to 2’-deoxy N 4 - hydroxycytidine triphosphate (2' deoxy NHC-TP) in cells, which can induce genotoxicity.
  • 2’ -deoxy N 4 -hydroxy cytidine diphosphate can be converted to 2' deoxy NHC-DP by RNR, which can be phosphorylated to 2' deoxy NHC-TP, that can be incorporated by cellular DNA polymerases, which in turn induces genotoxicity.
  • RNA virus When the compounds described herein are administered to a patient infected with an RNA virus, a portion of the drug is utilized for its intended purpose, i.e., inactivation of RNA viruses, and a portion is excreted, in its original form, or as one or more metabolites.
  • the deuterated and/or methylated compounds described herein will delay metabolism at the 3’-position, i.e., delay the conversion of the compounds described herein to deoxyribonucleotide analogs.
  • the compounds can be incorporated into the RNA of the RNA viruses, and all or most of the remaining compound that is not incorporated into the RNA of the RNA viruses is excreted or metabolized via a different pathway before being converted to the deoxyribonucleic acid analog, and incorporated by cellular DNA polymerases, which in turn reduces or avoids genotoxicity.
  • Ribonucleotide reductase also known as ribonucleoside diphosphate reductase (rNDP) is an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides.
  • This example shows an RNR assay to follow the production of deoxy-NHC-DP over time, and a cell culture assay to follow the production of deoxy-NHC, -MP, -DP, and -TP over time.
  • the RNR assay was conducted, according to the techniques described in Brignole et al., eLife 2018;7:e31502 doi: 10.7554/eLife.31502.
  • the mixtures were incubated at 37°C for 1hr in a Thermocycler, then heat inactivated at 95°C for 3 min in the Thermocycler.
  • the samples were diluted two hundred and fifty fold (250 X) and then subjected to LC-MS/MS analysis to follow the loss of rCDP, and the gain of 2' deoxy NHC-TP, over time.
  • Figures 1 A-D show the results in Figures 1 A-D, where Figure 1 A shows a CDP control, Figure IB shows the CDP reaction, Figure 1C shows the NHC-DP control, and Figure ID shows the NHC-DP reaction.
  • CTP/dCTP or NHC-DP/deoxy-NHC-DP were also detected.
  • ADP and AMP were detected in all of the samples, including the control sample.
  • ATP as a phosphate donor played the role in converting CDP to CTP and NHC-DP to NHC-TP.
  • a 12-well plate was seeded with 1 million vero cells/well. Either 10 ⁇ M or 50 ⁇ M NHC was added to each well, and the plate was incubated for 4 h at 37°C.
  • the NHC derivatives were measured in both supernatant and cell pellet using LC- MS/MS.
  • the LC-MS/MS instrument was a Thermo TSQ Quantiva, equipped with a Kinetex EVO-C18 (100 X 2.1 mm, 2.6 ⁇ m) column.
  • the buffers used for the liquid chromatography were as follows: For cell pellet extractions: A) 2 mM ammonium phosphate and 3 mM hexylamine, B) acetonitrile.
  • MS MRM mode, both positive and negative, monitoring for NHC, deoxy-NHC, and their respective monophosphates (-MP), diphosphates (-DP) and triphosphates (-TP).
  • CDP was converted to dCDP by RNR, with the ratio in the samples around 100:0.7 (CDP: dCDP), and 100:0.1 (CTP:dCTP), calculated by comparing peak areas.
  • NHC-DP was converted to deoxy-NHC-DP by RNR with a much lower efficiency than CDP to dCDP.
  • the ratio in the samples was around 100:0.1 (NHC- DP: deoxy-NHC-DP) and 100:0.009 (NHC-TP: deoxy-NHC-TP).
  • the conversion percentage could not be calculated, since a relatively large amount of CDP or NHC-DP was phosphorylated to CTP or NHC-TP by the reaction system, even in the control samples.
  • the toxicity of the compounds can be assessed in Vero, human PBM, CEM (human lymphoblastoid), MT-2, and HepG2 cells, as described previously (see Schinazi R.F., Sommadossi J.-P., Saalmann V., Cannon D.L., Xie M.-Y., Hart G.C., Smith G.A. & Hahn E.F. Antimicrob. Agents Chemother. 1990, 34, 1061-67). Cycloheximide can be included as positive cytotoxic control, and untreated cells exposed to solvent can be included as negative controls.
  • the cytotoxicity IC50 can be obtained from the concentration-response curve using the median effective method described previously (see Chou T.-C. & Talalay P. Adv. Enzyme Regul. 1984, 22, 27-55; Belen’kii M.S. & Schinazi R.F. Antiviral Res. 1994, 25, 1-11).
  • Mitochondrial Toxicity Assays in HepG2 Cells i) Effect of Compounds on Cell Growth and Lactic Acid Production: The effect on the growth of HepG2 cells can be determined by incubating cells in the presence of 0 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M and 100 ⁇ M drug. Cells (5 x 10 4 per well) can be plated into 12-well cell culture clusters in minimum essential medium with nonessential amino acids supplemented with 10% fetal bovine serum, 1% sodium pyruvate, and 1% penicillin/ streptomycin and incubated for 4 days at 37°C. At the end of the incubation period the cell number can be determined using a hemocytometer.
  • HepG2 cells from a stock culture can be diluted and plated in 12-well culture plates at 2.5 x 10 4 cells per well.
  • Various concentrations (0 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M and 100 ⁇ M) of compound can be added, and the cultures can be incubated at 37°C in a humidified 5% CO 2 atmosphere for 4 days.
  • the number of cells in each well can be determined and the culture medium collected.
  • the culture medium can then be filtered, and the lactic acid content in the medium determined using a colorimetric lactic acid assay (Sigma-Aldrich).
  • lactic acid product can be considered a marker for impaired mitochondrial function
  • elevated levels of lactic acid production detected in cells grown in the presence of test compounds indicates a drug- induced cytotoxic effect.
  • This assay can be used in all studies described in this application that determine the effect of compounds on mitochondrial DNA content.
  • low-passage- number HepG2 cells are seeded at 5,000 cells/well in collagen-coated 96-well plates. Test compounds are added to the medium to obtain final concentrations of 0 ⁇ M, 0.1 ⁇ M, 10 ⁇ M and 100 ⁇ M.
  • cellular nucleic acids can be prepared by using commercially available columns (RNeasy 96 kit; Qiagen). These kits co-purify RNA and DNA, and hence, total nucleic acids are eluted from the columns.
  • the mitochondrial cytochrome c oxidase subunit II (COXII) gene and the B-actin or rRNA gene can be amplified from 5 ⁇ l of the eluted nucleic acids using a multiplex Q-PCR protocol with suitable primers and probes for both target and reference amplifications.
  • COXII the following sense, probe and antisense primers can be used, respectively: 5'- TGCCCGCCATCATCCTA-3', 5'-tetrachloro-6-carboxyfluorescein- TCCTCATCGCCCTCCCATCCC-TAMRA-3' and 5'-
  • CGTCTGTTATGTAAAGGATGCGT-3' CGTCTGTTATGTAAAGGATGCGT-3'.
  • the sense, probe, and antisense primers are 5'- GCGCGGCTACAGCTTCA- 3', 5'-6-FAMCACCACGGCCGAGCGGGATAMRA-3' and 5'- TCTCCTTAATGTCACGCACGAT-3', respectively.
  • the primers and probes for the rRNA gene are commercially available from Applied Biosystems. Since equal amplification efficiencies are obtained for all genes, the comparative CT method can be used to investigate potential inhibition of mitochondrial DNA synthesis.
  • the comparative CT method uses arithmetic formulas in which the amount of target (COXII gene) is normalized to the amount of an endogenous reference (the ⁇ -actin or rRNA gene) and is relative to a calibrator (a control with no drug at day 7).
  • the arithmetic formula for this approach is given by 2- ⁇ CT, where ⁇ CT is (CT for average target test sample - CT for target control) - (CT for average reference test -CT for reference control) (see Johnson MR, K Wang, JB Smith, MJ Heslin, RB Diasio. Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. Anal. Biochem. 2000; 278:175-184).
  • a decrease in mitochondrial DNA content in cells grown in the presence of drug indicates mitochondrial toxicity.
  • HepG2 cells can be plated on 96 or 384 well tissue culture polystyrene plates. After 24 hr the cells can be dosed with test compound at a range of concentrations and incubated for 72 hr in medium supplemented with either galactose or glucose. Test compounds are said to cause mitochondrial toxicity if the cells grown in galactose-containing medium are more sensitive to the test compound than the cells grown in glucose-containing medium. Objective: To measure the sensitivity of HepG2 cells grown in medium containing either galactose or glucose to the test compound.
  • HepG2 human hepatocellular carcinoma cells are plated on 96 or 384-well tissue culture polystyrene plates containing either galactose or glucose containing medium supplemented with 10 % fetal bovine serum and antibiotics and incubated overnight.
  • Cell viability is measured using Hoechst staining and cell counting by a HCS reader.
  • mouse Neuro2A cells (American Type Culture Collection 131) can be used as a model system (see Ray AS, Hernandez-Santiago BI, Mathew IS, Murakami E, Bozeman C, Xie MY, Dutschman GE, Gullen E, Yang Z, Hurwitz S, Cheng YC, Chu CK, McClure H, Schinazi RF, Anderson KS. Mechanism of anti-human immunodeficiency virus activity of beta-D-6- cyclopropylamino-2’,3’-didehydro-2’,3’-dideoxyguanosine. Antimicrob. Agents Chemother. 2005, 49, 1994-2001).
  • concentrations necessary to inhibit cell growth by 50% can be measured using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyltetrazolium bromide dye- based assay, as described.
  • Perturbations in cellular lactic acid and mitochondrial DNA levels at defined concentrations of drug can be carried out as described above.
  • ddC and AZT can be used as control nucleoside analogs.
  • CFU-GM assays is carried out using a bilayer soft agar in the presence of 50 units/mL human recombinant granulocyte/macrophage colony- stimulating factor, while BFU-E assays used a ethylcellulose matrix containing 1 unit/mL erythropoietin (see Sommadossi JP, Carlisle R. Toxicity of 3’-azido-3’-deoxythymidine and 9-(1,3-dihydroxy-2-propoxymethyl) guanine for normal human hepatopoietic progenitor cells in vitro.
  • POLRMT In vitro human mitochondrial RNA polymerase
  • RNA nucleotide incorporation assays with POLRMT can be performed as previously described (Arnold et al. 2012). Briefly, 32 P-radiolabeled RNA primer (5’-UUUUGCCGCGCC) can be hybridized to 3 molar excess of the appropriate DNA template (5’-GGGAATGCANGGCGCGGC where position N can be replaced by A, T, or C). 125 nM of POLRMT can be incubated with 500 nM of 5’-radiolabled RNA/DNA hybrid, 10 mM MgCl 2 and 100 ⁇ M of the corresponding nucleoside triphosphate.
  • 100 ⁇ M of inhibitor can be added at the same time as 100 ⁇ M UTP. Incorporation can be allowed to proceed for 2 h at 30°C and reactions are stopped by the addition of 10 mM EDTA and formamide. Samples are visualized on 20% denaturing polyacrylamide gel. Data can be analyzed by normalizing the product fraction for each nucleoside triphosphate analog to that of the corresponding natural nucleoside triphosphate.
  • the protein concentration can be determined spectrophotometrically at 280 nm, with extinction coefficients of 234,420, and 71,894 M-1 cm-1 for the large and the small subunits of polymerase y, respectively.
  • Kinetic Analyses of Nucleotide Incorporation Pre-steady-state kinetic analyses can be performed to determine the catalytic efficiency of incorporation (k/K) for DNA polymerase ⁇ for nucleoside-TP and natural dNTP substrates. This allowed determination of the relative ability of this enzyme to incorporate modified analogs and predict toxicity.
  • the human polymerase y exonuclease activity can be studied by measuring the rate of formation of the cleavage products in the absence of dNTP.
  • the reaction can be initiated by adding MgCl 2 (2.5mM) to a pre-incubated mixture of polymerase ⁇ large subunit (40nM), small subunit (270nM), and 1,500nM chain-terminated template/primer in 50mM Tris-HCl, lOOmM NaCl, pH 7.8, and quenched with 0.3M EDTA at the designated time points.
  • nucleoside-triphosphate analog inhibits human DNA polymerases Alpha, Beta and Gamma and to calculate IC 50 values.
  • Human DNA Polymerase Alpha - Enzyme can be purchased from Chimerx (cat#1075) and assayed based on their recommendations with some modifications.
  • the 2’-Me-UTP was treated with Inorganic Pyrophosphatase (Sigma) to remove any pyrophosphate contamination.
  • a final concentration of 500 ⁇ M 2’-Me-UTP can be incubated with 1 mM DTT, 50 mM Tris, 50 mM NaCl, 6 mM MgCI 2 , and 1 unit of pyrophosphatase for 1 hour at 37°C followed by inactivation at 95°C for 10 minutes.
  • CAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAGC can be mixed with increasing concentrations of compound from 0 to 100 ⁇ M in 60 mM Tris-HCl (pH 8.0), 5 mM magnesium acetate, 0.3 mg/ml bovine serum albumin, 1 mM dithiothreitol, 0.1 mM spermine, 0.05 mM of each dCTP, dGTP, dTTP, dATP in a final reaction volume of 20 ⁇ l for 5 min at 37°C (all concentrations represent final concentrations after mixing). The reactions can be stopped by mixing with 0.3 M (final) EDTA.
  • Human DNA Polymerase Beta - Enzyme can be purchased from Chimerx (cat#1077) and assayed based on their recommendations with some modifications.
  • a mixture of 0.1 units of Human DNA Polymerase Beta and a 5 ’end radiolabeled 24nt DNA primer (5’- TCAGGTCCCTGTTCGGGCGCCACT) anneal to aa 48nt DNA template (5’- CAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAGC) can be mixed with increasing concentrations of compound from 0 to 100 ⁇ M in 50 mM Tris-HCl (pH 8.7), 10 mM KCl, 10 mM MgCI 2 , 0.4 mg/ml bovine serum albumin, 1 mM dithiothreitol, 15% (v/v) glycerol, and 0.05 mM of each dCTP, dGTP, dTTP, dATP in a final reaction volume of 20 ⁇ l for 5 min at 37
  • the reactions can be stopped by mixing with 0.3 M (final) EDTA. Products can be separated on a 20% polyacrylamide gel and quantitated on a Bio-Rad Molecular Imager FX. Results from the experiments can be fit to a dose response equation, (y min +((y max)-(y min)))/(l+(compound concentration)/IC 50 ) ⁇ slope) to determine IC 50 values using Graphpad Prism or SynergySoftware Kaleidagraph. Data can be normalized to controls..
  • Human DNA Polymerase Gamma - Enzyme can be purchased from Chimerx (cat#1076) and assayed based on their recommendations with some modifications.
  • a mixture of 0.625 units of Human DNA Polymerase Gamma and a 5’end radiolabeled 24nt DNA primer (5’-TCAGGTCCCTGTTCGGGCGCCACT) anneal to a 36nt DNA template (5’- TCTCTAGAAGTGGCGCCCGAACAGGGACCTGAAAGC) can be mixed with increasing concentrations of compound from 0 to 100 ⁇ M in 50 mM Tris-HCl (pH 7.8), 100 mM NaCl, 5 mM MgCI 2 , and 0.05 mM of each dCTP, dGTP, dTTP, dATP in a final reaction volume of 20 ⁇ l for 200 min at 37°C (all concentrations represent final concentrations after mixing).
  • the reactions can be stopped by mixing with 0.3 M (final) EDTA. Products can be separated on a 20% polyacrylamide gel and quantitated on a Bio-Rad Molecular Imager FX. Results from the experiments can be fit to a dose response equation, (y min +((y max)-(y min)))/(1+(compound concentration)/IC 50 ) ⁇ slope) to determine IC 50 values using Graphpad Prism or SynergySoftware Kaleidograph. Data can be normalized to controls.
  • HepG2 cells are obtained from the American Type Culture Collection (Rockville, MD), and are grown in 225 cm 2 tissue culture flasks in minimal essential medium supplemented with non-essential amino acids, 1% penicillin-streptomycin. The medium is renewed every three days, and the cells are subcultured once a week. After detachment of the adherent monolayer with a 10 minute exposure to 30 mL of trypsin-EDTA and three consecutive washes with medium, confluent HepG2 cells are seeded at a density of 2.5 x 10 6 cells per well in a 6-well plate and exposed to 10 ⁇ M of [ 3 H] labeled active compound (500 dpm/pmol) for the specified time periods.
  • the cells are maintained at 37°C under a 5% CO 2 atmosphere. At the selected time points, the cells are washed three times with ice-cold phosphate-buffered saline (PBS).
  • PBS ice-cold phosphate-buffered saline
  • Intracellular active compound and its respective metabolites are extracted by incubating the cell pellet overnight at -20°C with 60% methanol followed by extraction with an additional 20 pal of cold methanol for one hour in an ice bath. The extracts are then combined, dried under gentle filtered air flow and stored at -20°C until HPLC analysis.
  • Test compounds are incubated in PBM cells at 50 ⁇ M for 4 h at 37°C. Then the drug containing media is removed and the PBM cells are washed twice with PBS to remove extracellular drugs.
  • the intracellular drugs are extracted from 10 x 10 6 PBM cells using 1 mL 70% ice-cold methanol (containing 10 nM of the internal standard ddATP). Following precipitation, the samples are maintained at room temperature for 15 min followed by vortexing for 30 sec, and then stored 12 h at -20°C. The supernatant is then evaporated to dryness. Dry samples would be stored at -20°C until LC-MS/MS analysis. Prior to analysis, each sample is reconstituted in 100 ⁇ L mobile phase A, and centrifuged at 20,000 g to remove insoluble particulates.
  • the total run time is 33 min.
  • the flow rate is maintained at 50 ⁇ L/min and a 10 ⁇ L injection is used.
  • the autosampler and the column compartment are typically maintained at 4.5 and 30°C, respectively.
  • the first 3.5 min of the analysis is diverted to waste.
  • the mass spectrometer is operated in positive ionization mode with a spray voltage of 3.2 kV.
  • MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
  • IC 50 cytotoxic concentrations of drug required to reduce cell viability by 50%
  • MNTC maximum non-toxic concentrations
  • the resultant inhibitory effect of each test compound is calculated as a percentage of decrease in EV-71 CPE.
  • a monolayer of RD cells is prepared in a 96-well plates. The cells are then infected with 1 MOI of EV-71 (BrCr strain) followed by treatment with a single non-toxic dose of each compound in triplicate. The vehicle control wells are treated with 0.1% DMSO diluted in the working media. The plate is then incubated for 48 h at which time the virus-control wells produced detectable CPE.
  • an MTS assay is performed and effective compounds are chosen for further studies to identify the potency of the compounds and their concentration- dependent manner.
  • the dose-response antiviral activity of each compound is determined by a virus yield reduction assay method. Briefly, confluent monolayers of RD cells in 96-wells microplate are infected with 0.1 MOI of EV-71 followed by treatment with compounds. Effective compounds are further quantified and confirmed with a virus-yield-reduction assay using an optimized in house qRT-PCR to determine the EV-71 RNA copy number after 2 days post-treatment from collected supernatants. qRT-PCR is performed using the EV-71 specific probe/primer mix and qScript-Tough master mix (Quantibio, USA). Quantitative PCR measurement was performed using StepOnePlus real time PCR system (Roche, Germany) according to manufacturer’s protocol.
  • the median effective concentration (EC 50 ) and the concentration with 90% of inhibitory effect (EC 90 ) are calculated using GraphPad PRISM for Windows, version 5 (GraphPad Software Inc., San Diego, CA, 2005) as the means ⁇ standard deviation (SD) of the mean from triplicate assay from three independent experiments.
  • Anti-Chikungunya Activity can also be evaluated as outlined in “Anti-Chikungunya Viral Activities of Aplysiatoxin-Related Compounds from the Marine Cyanobacterium Trichodesmium erythraeum” Gupta, D. K.; Kaur, P.; Leong, S. T.; Tan, L. T.; Prinsep, M. R.; Chu, J J. H. Mar Drugs. Jan 2014; 12(1): 115-127; 10.3390/mdl2010115 and references cited therein.
  • a representative assay for determining the efficacy of the compounds described herein against the Mayaro virus, another representative Togaviridae virus, is disclosed in Cavalheiro et al., “Macrophages as target cells for Mayaro virus infection: involvement of reactive oxygen species in the inflammatory response during virus replication,” Anais da Academia Brasileira de Ciências (2016) 88(3): 1485-1499, (Annals of the Brazilian Academy of Sciences). The procedures are summarized below.
  • RAW 264.7 a mouse leukaemic macrophage cell line, and J774, a mouse reticulum sarcoma cell line
  • LGC RPMI- 1640 medium
  • FBS fetal bovine serum
  • Mouse peritoneal macrophages can be obtained from C57B1/6 animals by the intraperitoneal injection of 1 mL of sterile 3% thioglycollate. After 96 h, the peritoneal macrophages can be harvested, washed with RPMI and centrifuged at 1,500 rpm for five minutes.
  • the macrophages can be plated at a density of 2 x 10 6 cells/well in a 6-well plate with RPMI-1640 supplemented with 10% FBS and incubated at 37°C with 5% CO 2 . After 24 h, the plates can be washed with RPMI to remove non-adherent cells before the assays.
  • MAYV (ATCC VR 66, strain TR 4675) and SINV (AR339) can be propagated in BHK- 21 cells grown in a-Minimum Essential Medium ( ⁇ -MEM; Invitrogen Life Technologies) supplemented with 10% FBS.
  • the cells can be infected with a multiplicity of infection (MOI) of 0.1.
  • MOI multiplicity of infection
  • the culture media can be harvested and cell debris can be removed by centrifugation at 2,000 x g for 10 min and the supernatant can be stored at -80°C.
  • Virus stocks titers can be determined by plaque assay in BHK-21 cells.
  • Cells can be incubated with MAYV or SINV at a MOI of 1 (for RAW 264.7 and J774) or 5 (for primary peritoneal macrophages), for 1 h at 37°C in 5% CO2. Then, the medium containing the non-adsorbed virus can be removed, the cells can be washed with serum-free medium and cultured in RPMI supplemented with 5% FBS, at 37°C in 5% CO2. After the desired periods of infection, conditioned media can be collected for virus titration, LDH assay and cytokine quantification. Cellular extracts can be used for MTT and flow cytometry assays. Virus inactivated by heating at 65°C for 30 min can be used as control.
  • cells can be treated with 10 mM N-acetyl-L-cysteine (NAC; Sigma- Aldrich) or 50 ⁇ M apocynin (Sigma-Aldrich) for 15h after infection with MAYV.
  • NAC N-acetyl-L-cysteine
  • apocynin Sigma-Aldrich
  • BHK-21 cells can be seeded, for example, at a density of 1.25 x 10 5 cells per well in 12- wells plates and incubated at 37°C overnight.
  • Ten-fold serial dilutions of the virus samples can be prepared in a-MEM and incubated with the cells for 1 h at 37°C (0.2 mL per well).
  • 2 mL of 1% carboxymethylcellulose (w/v) (Sigma- Aldrich) in a-MEM supplemented with 2% FBS can be layered onto the infected monolayers and the cells can be incubated at 37°C for 30 h or 48 h, for SINV or MAYV, respectively. Plaques can be visualized by staining the monolayer with 1 mL 1% crystal violet in 20% ethanol.
  • MTT 3-(4,5- dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide
  • LDH lactate dehydrogenase release assays.
  • MTT assay cells can be incubated with 0.5 mL 0.5 mg/mL MTT (USB Corporation) in PBS solution for 90 min at 37°C. Then, unreacted dye can be discarded and formazan crystals can be An Acad Bras Cienc (2016) 88 (3) 1488 Mariana G. Cavalheiro et al. solubilized in 0.04 M HCl solution in isopropanol (1 mL per well).
  • Lactate dehydrogenase (LDH) release from infected macrophages can be determined by using an LDH detection kit (Promega CytoTox 96 assay kit). The procedures can be performed according to manufacturer’s instructions.
  • Flow cytometry analysis can be performed to assess the frequency of MAYV- or SINV- infected cells by detecting intracellular viral antigens.
  • cells can be washed with PBS, detached by scraping, harvested and fixed in 4% formaldehyde in PBS at room temperature for 15 min.
  • cells can be permeabilized with 0.1% saponin in PBS and incubated with blocking solution (PBS supplemented with 2% FBS and 0.1% bovine serum albumin) for 20 min, at room temperature. Then, cells can be incubated for 1 h with mouse anti-Eastem Equine Encephalitis virus monoclonal antibody (Chemicon International, Millipore), which reacts with an El epitope shared by all alphaviruses.
  • cells can be washed and stained with anti-mouse IgG conjugated to Alexa Fluor 488 (Invitrogen) for 30 min. The percentage of infected cells can be analyzed by FACScan Flow Cytometer and CellQuest software (Becton Dickinson).
  • Apoptosis/necrosis after infection can be quantified by a double staining method using The Vybrant Apoptosis Assay Kit#2 (Molecular Probes). After the infection period, RAW 264.7 cells can be washed with PBS, detached by scraping, harvested and stained with Annexin V Alexa Fluor 488 (0.5 ⁇ g/ mL) and propidium iodide (PI, 0.25 ⁇ g/mL). To further characterize MAYV-induced cell death, the activity of caspases 3 and 7 can be measured using the MuseTM Caspase-3/7 Kit (Millipore) adapted to flow cytometry.
  • Cells can be washed with PBS, detached by scraping, harvested and incubated with MuseTM Caspase-3/7 Reagent 1:8 and MuseTM Caspase 7-AAD, according to the manufacturer's protocol.
  • MuseTM Caspase-3/7 Reagent 1:8 and MuseTM Caspase 7-AAD according to the manufacturer's protocol.
  • the percentage of apoptotic and necrotic cells can be analyzed by FACScan Flow Cytometer using the CellQuest software (Bectan Dickinson). UV radiated cells and cells subjected to a freeze-thaw procedure can be used as controls.
  • ROS Reactive Oxygen Species
  • ROS reactive oxygen species
  • cytokines concentrations in the conditioned medium of macrophage cultures can be determined by ELISA. TNF concentration can be quantified using the Standard ELISA Development kit (PeproTech), according to the manufacturer’s protocol.
  • Yellow Fever Virus (YFV) Antiviral Activity Assay Primary assay for antiviral activity
  • a monolayer of human Rhabdomyosarcoma (RD) cells will be grown in 96-well plate in MEM containing 2% inactivated FBS.
  • the plate will then be incubated at 37°C with 5% CO 2 for 72 hours.
  • the assay will be conducted in triplicate for each concentration of each compound. After three days, the plate will be viewed under the microscope and the degree of cytopathic effect (CPE) as measure of virus replication inhibition will be expressed as the percent yield of virus control.
  • CPE degree of cytopathic effect
  • Antiviral activity of each compound will be determined by measuring the reduction in the number of YFV infectious foci in RD cells following treatment with increasing concentrations of each compound. Briefly, infected RD cells which will be treated with different concentrations of each compound will be incubated for 2 days post infection using conditioned- growth medium supplemented with 2% FBS and 1.5% carboxymethyl cellulose (CMC). Antiviral activity of each compound will be determined after visualizing and counting viral foci. The number of YFV foci will be counted using Elispot machine and the virus titer will be expressed as Foci Forming-Unit (FFU).
  • FFU Foci Forming-Unit
  • Graph Pad Prism for Windows, Version 5 (Graph Pad Software Inc., San Diego, CA, 2005) will be used to determine the half maximal effective concentration EC 50 values and also EC 90 of each effective compound. All EC 50 and EC 90 values will be calculated as the means ⁇ standard error of the mean (SEM) from triplicate assay from three independent experiments.
  • the assay relates to human and Dengue virus polymerase, where putative compounds can be tested against the enzymes, preferably in duplicate, over a range of concentrations, such as from 0.8 mM to 100 mM.
  • the compounds can also be run alongside a control (no inhibitor), a solvent dilution (0.016% to 2% DMSO) and a reference inhibitor.
  • Dengue virus (DENV) NS5 possesses methyltransferase (MTase) activity at its N-terminal amino acid sequence and is responsible for formation of a type 1 cap structure, m7GpppAm2'-O in the viral genomic RNA.
  • MTase methyltransferase
  • Optimal in vitro conditions for DENV22'-O-MTase activity can be characterized using purified recombinant protein and a short biotinylated GTP-capped RNA template.
  • a GTP-binding pocket present at the N-terminal of DENV2 MTase can be previously postulated to be the cap-binding site. This assay allows rapid and highly sensitive detection of 2'-O-MTase activity, and can be readily adapted for high-throughput screening for inhibitory compounds.
  • Compounds can exhibit anti-norovirus activity by inhibiting norovirus polymerase and/or helicase, by inhibiting other enzymes needed in the replication cycle, or by other pathways.
  • Norovirus replicons and hepatitis C replicons require viral helicase, protease, and polymerase to be functional in order for replication of the replicon to occur.
  • an in vitro cell culture infectivity assay has been reported utilizing Norovirus genogroup I and II inoculums (Straub, T. M. et al. (2007) Emerg. Infect. Dis. 13(3):396-403). This assay is performed in a rotating-wall bioreactor utilizing small intestinal epithelial cells on microcarrier beads. The infectivity assay may be useful for screening entry inhibitors.
  • RT-PCR reverse transcription-polymerase chain reaction
  • Viruses ZIKVPRVABC59 strain (NCBI accession KU501215) was obtained from the Centers for Diseases Control and Prevention. Virus stocks were generated on C6/36 or Vero cells and viral titers are determined by endpoint titration in Vero (African Green monkey kidney) or human cells, including neuroblastoma (U251), and hepatoblastoma (Huh7). DENV stocks (kindly provided by Dr. Guey Chuen Pemg (Emory University & National Cheng Kung University, Taiwan) were generated in Vero or Baby Hamster Kidney cells (BHK) (Clark et al., 2016).
  • Cytopathic-reduction assay for ZIKV or DENV For the cytopathic-reduction assay, cells (Vero, U251 or Huh7) are seeded in 96-well plates at 1x10 4 cells/well and incubated overnight. The next day, culture medium containing 50% cell culture infectious doses of ZIKV or DENV (tested in Vero or BHK cells) are added after which 2-fold serial dilutions of the compounds are added.
  • CPE Cell cytopathic effect
  • Focus formation assay For the focus formation assay (FFA), Vero cells are routinely seeded in 96-well plates at 1.5x10 4 cells/well and incubated overnight. Next, culture medium containing 70-100 focus forming units of ZIKV or DENV (serotypes 1-4) plus 2-fold serial dilutions of the compounds are added to the cells and incubated for 2 h followed by the addition of overlay methylcellulose medium. Following 2-3 days of incubation, foci are stained using anti-Flavivirus group antigen (4G2, Millipore), followed by HRP-anti-mouse IgG and TrueBlue substrate, and imaged using CTL-Immunospot S6 Micro Analyzer (Priyamvada et al., 2016).
  • FFA focus formation assay
  • RNA are reverse transcribed into cDNA and amplified in a one-step RT-PCR multiplex reaction with LightCycler 480 RNA Master Hydrolysis Probe (Roche, Indianapolis, IN) using highly conserved sequences complementary to a 76 bp fragment from the ZIKV envelope gene as previously described by Lanciotti (Lanciotti et al., 2008), and an endogenous control (TaqMan Ribosomal RNA Control or beta globin reagents; Applied Biosystems) by using the LightCycler 480 Instrument II (Roche).
  • LightCycler 480 RNA Master Hydrolysis Probe (Roche, Indianapolis, IN) using highly conserved sequences complementary to a 76 bp fragment from the ZIKV envelope gene as previously described by Lanciotti (Lanciotti et al., 2008), and an endogenous control (TaqMan Ribosomal RNA Control or beta globin reagents; Applied Biosystems) by using the LightCycler
  • oligonucleotides primers and probes serotype-specific that rapidly detects all four serotypes in a fourplex RT-PCR assay (Johnoson et al., 2005).
  • percent inhibition and EC 50 value are determined using CalcuSyn software (Biosoft).
  • active compounds for use in treating ZIKV or DENV have sub- ⁇ M concentrations for hitto lead development, with cell selectivity index (SI) ⁇ 100.
  • Hit compounds that demonstrate antiviral potency with no apparent cytotoxicity can be selected for drug-drug combinations with compounds that exhibit different mechanism of action, including viral entry and host inhibitors, among others; These combinations can result in synergistic effects and optimal low doses to rapidly eliminate ZIKV or DENV from infected individuals.
  • DENV2 replicon-harboring baby hamster kidney (BHK) cells are exposed to test compounds at concentrations varying from 0.2 to 20 ⁇ M to assessment of antiviral activity. Renilla luciferase levels (Promega) are quantified 48 hours after test compounds addition to determine the levels of replication inhibition (EC 50 , ⁇ M).
  • Human lung carcinoma cells can be used for the primary antiviral assays and can be obtained from American Type Culture Collection (ATCC, Rockville, Md., USA). The cells can be passed in minimal essential medium (MEM with 0.15% NaCHO 3 , Hyclone Laboratories, Logan, Utah, USA) supplemented with 10% fetal bovine serum. When evaluating compounds for efficacy, the serum can be reduced to a final concentration of 2% and the medium can contain gentamicin (Sigma-Aldrich, St. Louis, Mo.) at 50 ⁇ g/mL. Since the MERS-Co virus did not produce detectable virus cytopathic effects, virus replication in A549 cells can be detected by titering virus supernatant fluids from infected, compound- treated A549 cells in Vero 76 cells.
  • MEM minimal essential medium
  • gentamicin Sigma-Aldrich, St. Louis, Mo.
  • Vero 76 cells can be obtained from ATCC and can be routinely passed in MEM with 0.15% NaCHO 3 supplemented with 5% fetal bovine serum. When evaluating compounds, the serum can be reduced to a final concentration of 2% and supplemented with 50 ⁇ g/mL of gentamicin.
  • MERS-CoV Middle Eastern coronavirus strain
  • Infergen® interferon alfacon-1, a recombinant non-naturally occurring type-I interferon (Blatt, L., et al., J. Interferon Cytokine Res. (1996) 16(7):489-499 and Alberti, A., BioDrugs (1999) 12(5):343-357) can be used as the positive control drug in all antiviral assays.
  • hifergen 0.03 ng/mL.
  • Compound can be added first to 96 well plates of confluent A549 cells followed within 5 mins by virus. Each test compound dilution can be evaluated for inhibition in triplicate. After plating, the plates can be incubated at 37° C. for 4 d. The plates can then be frozen at -80° C.
  • Infectious virus yields from each well from the antiviral assay can be determined. Each plate from an antiviral assays can be thawed. Samples wells at each compound concentration tested can be pooled and titered for infectious virus by CPE assay in Vero 76 cells. The wells can be scored for CPE and virus titers calculated. A 90% reduction in virus yield can then be calculated by regression analysis. This represented a one log 1 0 inhibition in titer when compared to untreated virus controls.
  • VEEV Venezuelan equine encephalitis virus
  • 96-well plates of HeLa-Ohio cells can be prepared and incubated overnight. The plates can be seeded at 4 X 10 4 cells per well, which yields 90-100% confluent monolayers in each well after overnight incubation.
  • the test compounds in DMSO can be started at a concentration of 100 ⁇ M. 8-fold serial dilutions in MEM medium with 0.1% DMSO, 0% FBS, and 50 ⁇ g/mL gentamicin with the test compound concentrations being prepared. To 5 test wells on the 96- well plate can be added 100 ⁇ L of each concentration and the plate can be incubated at 37° C +5% CO 2 for 2 h or 18 h.
  • the median effective concentration (EC 50 ) and the concentration with 90% of inhibitory effect (EC 90 ) were calculated using GraphPad PRISM for Mac, version 7 (GraphPad Software Inc., San Diego, CA, 2005) and reported as the mean ⁇ standard deviation (SD).
  • An inhibitor triphosphate can interfere with RNA synthesis.
  • An RNA polymerase is an enzyme that synthesizes RNA from a DNA template. When a growing RNA chain comes into contact with an RNA polymerase and a naturally-occurring nucleoside triphosphate, the RNA chain is extended. However, when an unnatural inhibitor triphosphate is present, there is an error when the RNA polymerase seeks to add the inhibitor triphosphate to the growing RNA chain.
  • the complex can be buffered using 25mM Tris-HCl (pH 8). To this buffered solution of the RdRP complex can be added 50 ⁇ M of a 17-mer RNA primer and 1 ⁇ M ofa43-merRNA template, and the solution can be incubated on ice for around 15 minutes.
  • 0.1 ⁇ M hot GTP can be added, followed by addition of NTP mixtures (50 mM ATP, CTP and TTP; 25 mM GTP) to provide the nucleoside triphosphates needed for RNA synthesis. Then, either control (water) or an inhibitor triphosphate can be added.
  • NHC reduces the number of colony formed by about 50% compared with NHC.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Communicable Diseases (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des analogues de N4-hydroxycytidine (NHC) deutériée et/ou méthylée, avec une deutération à l'une ou aux deux positions 2'et 3'sur la fraction ribose et/ou une méthylation des positions 3' sur la fraction ribose, des compositions pharmaceutiques comprenant un ou plusieurs de ces composés et, éventuellement, au moins un agent thérapeutique supplémentaire, ainsi que des méthodes de traitement ou de prévention d'infections provoquées par des virus à ARN, de guérison d'une infection par un virus à ARN ou de réduction de l'activité biologique d'un virus à ARN. Les virus à ARN représentatifs comprennent, mais sans s'y limiter, les Coronaviridae, tels que le MERSr-CoV, le SARS-CoV-1, le SARS-CoV-2, le HCoV-OC43, le HCoV-229E, le HCoV-NL63 et le HCoV-HKU1, les Clanaviridae, les Hepeviridae, les norovirus, le Zika, la dengue, la fièvre de Mayaro, la grippe A et B, le virus Parainfluenza, le virus de l'hépatite c, le rinovirus, les virus transmis par les tiques, le virus Ebola, la fièvre de Lassa, le VRS, les adénovirus, les entérovirus, les métapneumovirus, les encéphalites équines de l'Est, de l'Ouest et vénézuelienne (EEE, WEE et VEE, respectivement), et le Chikungunya (CHIK).
PCT/US2022/031381 2021-05-27 2022-05-27 Nouveaux agents viraux anti-arn universels Ceased WO2022251663A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/562,911 US20240262857A1 (en) 2021-05-27 2022-05-27 Novel universal anti-rna virus agents

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163193951P 2021-05-27 2021-05-27
US63/193,951 2021-05-27

Publications (2)

Publication Number Publication Date
WO2022251663A2 true WO2022251663A2 (fr) 2022-12-01
WO2022251663A3 WO2022251663A3 (fr) 2022-12-29

Family

ID=84230314

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/031381 Ceased WO2022251663A2 (fr) 2021-05-27 2022-05-27 Nouveaux agents viraux anti-arn universels

Country Status (2)

Country Link
US (1) US20240262857A1 (fr)
WO (1) WO2022251663A2 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11660307B2 (en) 2020-01-27 2023-05-30 Gilead Sciences, Inc. Methods for treating SARS CoV-2 infections
CN116217644A (zh) * 2023-04-24 2023-06-06 南京颐媛生物医学研究院有限公司 抗冠状病毒核糖核苷类化合物及其制备方法和应用
US11701372B2 (en) 2020-04-06 2023-07-18 Gilead Sciences, Inc. Inhalation formulations of 1'-cyano substituted carba-nucleoside analogs
WO2023142803A1 (fr) * 2022-01-28 2023-08-03 北京恩泰伟医药科技有限公司 Composé antiviral et son utilisation
US11780844B2 (en) 2022-03-02 2023-10-10 Gilead Sciences, Inc. Compounds and methods for treatment of viral infections
US11814406B2 (en) 2020-08-27 2023-11-14 Gilead Sciences, Inc. Compounds and methods for treatment of viral infections
US11903953B2 (en) 2020-05-29 2024-02-20 Gilead Sciences, Inc. Remdesivir treatment methods
US11939347B2 (en) 2020-06-24 2024-03-26 Gilead Sciences, Inc. 1′-cyano nucleoside analogs and uses thereof
US12012431B2 (en) 2020-03-12 2024-06-18 Gilead Sciences, Inc. Methods of preparing 1′-cyano nucleosides
WO2024125604A1 (fr) * 2022-12-16 2024-06-20 Suzhou Spring-Sea Bio-Pharmaceuticals Co. Ltd. Dérivés diesters de n4-hydroxycytidine et leur utilisation
US12357577B1 (en) 2024-02-02 2025-07-15 Gilead Sciences, Inc. Pharmaceutical formulations and uses thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1572097A4 (fr) * 2002-09-30 2010-02-17 Smithkline Beecham Corp Derives nucleosidiques servant au traitement d'une infection par le virus de l'hepatite c
US6943249B2 (en) * 2003-03-17 2005-09-13 Ash Stevens, Inc. Methods for isolating crystalline Form I of 5-azacytidine
JP7381190B2 (ja) * 2014-12-26 2023-11-15 エモリー・ユニバーシテイ Ｎ４－ヒドロキシシチジン及び誘導体並びにそれに関連する抗ウイルス用途
WO2020206289A1 (fr) * 2019-04-05 2020-10-08 Prelude Therapeutics, Incorporated Inhibiteurs sélectifs de la protéine arginine méthyltransférase 5
MX2023011870A (es) * 2021-04-09 2023-12-07 Univ Emory Análogos de nucleósidos y de nucleótidos como agentes antivirales para coronavirus y otros virus.

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11660307B2 (en) 2020-01-27 2023-05-30 Gilead Sciences, Inc. Methods for treating SARS CoV-2 infections
US12012431B2 (en) 2020-03-12 2024-06-18 Gilead Sciences, Inc. Methods of preparing 1′-cyano nucleosides
US11701372B2 (en) 2020-04-06 2023-07-18 Gilead Sciences, Inc. Inhalation formulations of 1'-cyano substituted carba-nucleoside analogs
US11975012B2 (en) 2020-05-29 2024-05-07 Gilead Sciences, Inc. Remdesivir treatment methods
US11903953B2 (en) 2020-05-29 2024-02-20 Gilead Sciences, Inc. Remdesivir treatment methods
US11939347B2 (en) 2020-06-24 2024-03-26 Gilead Sciences, Inc. 1′-cyano nucleoside analogs and uses thereof
US12404289B2 (en) 2020-06-24 2025-09-02 Gilead Sciences, Inc. 1′-cyano nucleoside analogs and uses thereof
US12297226B2 (en) 2020-08-27 2025-05-13 Gilead Sciences, Inc. Compounds and methods for treatment of viral infections
US11814406B2 (en) 2020-08-27 2023-11-14 Gilead Sciences, Inc. Compounds and methods for treatment of viral infections
US11926645B2 (en) 2020-08-27 2024-03-12 Gilead Sciences, Inc. Compounds and methods for treatment of viral infections
WO2023142803A1 (fr) * 2022-01-28 2023-08-03 北京恩泰伟医药科技有限公司 Composé antiviral et son utilisation
US11780844B2 (en) 2022-03-02 2023-10-10 Gilead Sciences, Inc. Compounds and methods for treatment of viral infections
US11851438B2 (en) 2022-03-02 2023-12-26 Gilead Sciences, Inc. 1′-cyano nucleoside analogs and methods for treatment of viral infections
US11845755B2 (en) 2022-03-02 2023-12-19 Gilead Sciences, Inc. Compounds and methods for treatment of viral infections
US12180217B2 (en) 2022-03-02 2024-12-31 Gilead Sciences, Inc. Compounds and methods for treatment of viral infections
US12448383B2 (en) 2022-03-02 2025-10-21 Gilead Sciences, Inc. Compounds and methods for treatment of viral infections
WO2024125604A1 (fr) * 2022-12-16 2024-06-20 Suzhou Spring-Sea Bio-Pharmaceuticals Co. Ltd. Dérivés diesters de n4-hydroxycytidine et leur utilisation
CN116217644B (zh) * 2023-04-24 2023-08-08 南京颐媛生物医学研究院有限公司 抗冠状病毒核糖核苷类化合物及其制备方法和应用
CN116217644A (zh) * 2023-04-24 2023-06-06 南京颐媛生物医学研究院有限公司 抗冠状病毒核糖核苷类化合物及其制备方法和应用
US12357577B1 (en) 2024-02-02 2025-07-15 Gilead Sciences, Inc. Pharmaceutical formulations and uses thereof

Also Published As

Publication number Publication date
WO2022251663A3 (fr) 2022-12-29
US20240262857A1 (en) 2024-08-08

Similar Documents

Publication Publication Date Title
US20240262857A1 (en) Novel universal anti-rna virus agents
US20240238323A1 (en) Modified nucleosides and nucleotides analogs as antiviral agents for corona and other viruses
US12239655B2 (en) Combined modalities for nucleosides and/or NADPH oxidase (NOX) inhibitors as myeloid-specific antiviral agents
US20240216414A1 (en) Antiviral Agents and Nucleoside Analogs for Treatment of Zika Virus
CN107709344B (zh) 用于治疗黄病毒科病毒和癌症的核苷类似物
AU2020289560A1 (en) Peptidomimetics for the treatment of coronavirus and picornavirus infections
JP7633394B2 (ja) リン脂質化合物及びその使用
CN104884462A (zh) 用于治疗病毒感染和癌症的嘧啶核苷及其单磷酸酯前药
CN1478483A (zh) 用于治疗乙型肝炎的β-L-2'-脱氧-核苷
US20170334941A1 (en) 2',2'-dihalo nucleoside analogs for treatment of the flaviviridae family of viruses and cancer
US20170037078A1 (en) 2' -disubstituted nucleoside analogs for treatment of the flaviviridae family of viruses and cancer
CN1329618A (zh) 具有抗乙型肝炎病毒活性的核苷
CN117769427A (zh) 作为冠状病毒和其他病毒的抗病毒剂的修饰核苷和核苷酸类似物

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 18562911

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22812277

Country of ref document: EP

Kind code of ref document: A2